Protein and peptide tags with increased rate of spontaneous isopeptide bond formation and uses thereof
阅读说明：本技术 自发性异肽键形成速率提高的蛋白质和肽标签及其用途 (Protein and peptide tags with increased rate of spontaneous isopeptide bond formation and uses thereof ) 是由 M·豪沃思 A·基布尔 于 2018-04-24 设计创作，主要内容包括：本发明涉及包含肽标签(肽)和多肽(蛋白质)的两部分接头,其能够自发形成异肽键,特别是其中：a)所述肽包含SEQ ID NO：1所示的氨基酸序列,其中：(i)在位置1处的X是精氨酸或无氨基酸；(ii)在位置2处的X是甘氨酸或无氨基酸；(iii)在位置5处的X是组氨酸或苏氨酸；(iv)在位置11处的X是丙氨酸、甘氨酸或缬氨酸；以及(v)在位置14处的X是精氨酸或赖氨酸,其中当在位置1处的X无氨基酸时,在位置2处的X无氨基酸；并且b)所述多肽包含：i)SEQ ID NO：2所示的氨基酸序列；ii)包含SEQ ID NO：101所示的氨基酸序列的(i)的一部分；iii)与SEQ ID NO：2所示的序列具有至少80％的序列同一性的氨基酸序列,其中所述氨基酸序列包含在位置34处的赖氨酸、在位置80处的谷氨酸和以下中的一个或多个：1)在位置5处的苏氨酸；2)在位置16处的脯氨酸；3)在位置40处的精氨酸；4)在位置65处的组氨酸；5)在位置92处的脯氨酸；6)在位置100处的天冬氨酸；7)在位置108处的谷氨酸；8)在位置116处的苏氨酸,其中指定的氨基酸残基位于与SEQ ID NO：2中的位置等同的位置；或iv)包含与SEQ ID NO：101所示序列具有至少80％的序列同一性的氨基酸序列的(iii)的一部分,其中所述氨基酸序列在位置10处包含赖氨酸,在位置56处包含谷氨酸和以下中的一个或多个：1)在位置16处的精氨酸；2)在位置41处的组氨酸；3)在位置68处的脯氨酸；和4)在位置76处的天冬氨酸,其中指定的氨基酸残基位于与SEQ ID NO：101中的位置等同的位置,并且其中所述肽和多肽能够在SEQ ID NO：1的位置10处的天冬氨酸残基和SEQ ID NO：2的位置34处或SEQ ID NO：101的位置10处的赖氨酸残基之间自发形成异肽键。(The present invention relates to a two-part linker comprising a peptide tag (peptide) and a polypeptide (protein), which is capable of spontaneously forming isopeptide bonds, in particular wherein: a) the peptide comprises SEQ ID NO:1, wherein: (i) x at position 1 is arginine or no amino acid; (ii) x at position 2 is glycine or no amino acid; (iii) x at position 5 is histidine or threonine; (iv) x at position 11 is alanine, glycine, or valine; and (v) X at position 14 is arginine or lysine, wherein X at position 2 is free of amino acids when X at position 1 is free of amino acids; and b) the polypeptide comprises: i) SEQ ID NO: 2; ii) comprises SEQ ID NO: 101, or (ii) a portion of (i) the amino acid sequence set forth in seq id no; iii) and SEQ ID NO: 2, wherein the amino acid sequence comprises a lysine at position 34, a glutamic acid at position 80, and one or more of: 1) threonine at position 5; 2) a proline at position 16; 3) arginine at position 40; 4) histidine at position 65; 5) a proline at position 92; 6) aspartic acid at position 100; 7) a glutamic acid at position 108; 8) threonine at position 116, wherein the specified amino acid residue is located at a position that is identical to SEQ ID NO: 2, the positions in 2 are equivalent; or iv) comprises a sequence identical to SEQ ID NO: 101, wherein the amino acid sequence comprises a lysine at position 10, a glutamic acid at position 56, and one or more of the following: 1) arginine at position 16; 2) histidine at position 41; 3) a proline at position 68; and 4) aspartic acid at position 76, wherein the specified amino acid residue is located at a position identical to SEQ ID NO: 101, and wherein the peptides and polypeptides are capable of binding at a position equivalent to that in SEQ ID NO:1 and an aspartic acid residue at position 10 of SEQ ID NO: 2 or at position 34 of SEQ ID NO: 101 between lysine residues at position 10 of the peptide bond.)
1. A two-part linker comprising a peptide and a polypeptide, wherein:
a) the peptide comprises SEQ ID NO:1, wherein:
(i) x at position 1 is arginine or no amino acid;
(ii) x at position 2 is glycine or no amino acid;
(iii) x at position 5 is histidine or threonine, preferably histidine;
(iv) x at position 11 is alanine, glycine or valine, preferably alanine; and
(v) x at position 14 is arginine or lysine, preferably arginine,
wherein when X at position 1 is free of an amino acid, X at position 2 is free of an amino acid; and is
b) The polypeptide comprises:
i) SEQ ID NO: 2;
ii) comprises SEQ ID NO: 101, or (ii) a portion of (i) the amino acid sequence set forth in seq id no;
iii) and SEQ ID NO: 2, wherein the amino acid sequence comprises a lysine at position 34, a glutamic acid at position 80, and one or more of:
1) threonine at position 5;
2) a proline at position 16;
3) arginine at position 40;
4) histidine at position 65;
5) a proline at position 92;
6) aspartic acid at position 100;
7) a glutamic acid at position 108; and
8) a threonine at the position 116 of the amino acid sequence,
wherein the specified amino acid residues are located at positions corresponding to SEQ ID NO: 2, the positions in 2 are equivalent; or
iv) comprises a sequence identical to SEQ ID NO: 101, wherein the amino acid sequence comprises a lysine at position 10, a glutamic acid at position 56, and one or more of:
1) arginine at position 16;
2) histidine at position 41;
3) a proline at position 68; and
4) (iii) an aspartic acid at position 76,
wherein the specified amino acid residues are located at positions corresponding to SEQ ID NO: 101 at a position equivalent to the position in the first embodiment,
and wherein the peptides and polypeptides are capable of binding at the amino acid sequence of SEQ ID NO:1 and an aspartic acid residue at position 10 of SEQ ID NO: 2 or at position 34 of SEQ id no: 101 between lysine residues at position 10 of the peptide bond.
2. The two-part linker according to claim 1, wherein the peptide comprises one or more of:
1) histidine at position 5;
2) alanine at position 11; and
3) (ii) an arginine at position 14, wherein,
wherein the specified amino acid residues are located at positions corresponding to SEQ ID NO: 2, are equivalent positions.
3. The two-part linker of claim 1 or claim 2, wherein the peptide comprises SEQ ID NO: 3-5, preferably the amino acid sequence shown in SEQ ID NO: 5.
4. The two-part linker according to any one of claims 1 to 3, wherein the polypeptide comprises an amino acid sequence identical to SEQ ID NO: 2, wherein the amino acid sequence comprises a lysine at position 34, a glutamic acid at position 80, and all of:
1) threonine at position 5;
2) a proline at position 16;
3) arginine at position 40;
4) histidine at position 65;
5) a glutamic acid at position 108; and
6) a threonine at the position 116 of the amino acid sequence,
wherein the specified amino acid residues are located at positions corresponding to SEQ ID NO: 2, are equivalent positions.
5. The two-part linker according to any one of claims 1 to 3, wherein the polypeptide comprises a sequence identical to SEQ ID NO: 2, wherein the amino acid sequence comprises a lysine at position 34, a glutamic acid at position 80, and all of:
1) threonine at position 5;
2) a proline at position 16;
3) arginine at position 40;
4) histidine at position 65;
5) a proline at position 92;
6) a glutamic acid at position 108; and
7) a threonine at the position 116 of the amino acid sequence,
wherein the specified amino acid residues are located at positions corresponding to SEQ ID NO: 2, are equivalent positions.
6. The two-part linker according to any one of claims 1 to 3, wherein the polypeptide comprises an amino acid sequence identical to SEQ ID NO: 2, wherein the amino acid sequence comprises a lysine at position 34, a glutamic acid at position 80, and all of:
1) threonine at position 5;
2) a proline at position 16;
3) arginine at position 40;
4) histidine at position 65;
5) a proline at position 92;
6) aspartic acid at position 100;
7) a glutamic acid at position 108; and
8) a threonine at the position 116 of the amino acid sequence,
wherein the specified amino acid residues are located at positions corresponding to SEQ ID NO: 2, are equivalent positions.
7. The two-part linker according to any one of claims 1 to 6, wherein the polypeptide comprises one or more of:
1) glycine at position 12; and
2) a threonine at the position 22 of the amino acid sequence,
wherein the specified amino acid residues are located at positions corresponding to SEQ ID NO: 2, are equivalent positions.
8. The two-part linker according to any one of claims 1 to 7, wherein the peptide and/or the polypeptide is conjugated to a nucleic acid molecule, a protein, a peptide, a small molecule organic compound, a fluorophore, a metal-ligand complex, a polysaccharide, a nanoparticle, a nanotube, a polymer, a cell, a virus-like particle, or a combination thereof.
9. The two-part linker according to any one of claims 1 to 7, wherein the peptide and/or polypeptide is immobilized on a solid substrate.
10. A peptide comprising SEQ ID NO:1, wherein:
(i) x at position 1 is arginine or no amino acid;
(ii) x at position 2 is glycine or no amino acid;
(iii) x at position 5 is histidine or threonine, preferably histidine;
(iv) x at position 11 is alanine, glycine or valine, preferably alanine; and
(v) x at position 14 is arginine or lysine, preferably arginine,
wherein when X at position 1 is free of an amino acid, X at position 2 is free of an amino acid;
and wherein the peptide is capable of hybridizing to a polypeptide comprising SEQ ID NO: 2, wherein the isopeptide bond is formed at the amino acid sequence shown in SEQ ID NO:1 and an aspartic acid residue at position 10 of SEQ ID NO: 2 between lysine residues at position 34.
11. The peptide according to claim 10, wherein the peptide is defined according to any one of claims 2, 3, 8 or 9.
12. A polypeptide, comprising:
i) SEQ ID NO: 2; or
ii) comprises the sequence as set forth in SEQ ID NO: 101, or (ii) a portion of (i) the amino acid sequence set forth in seq id no;
iii) and SEQ ID NO: 2, wherein the amino acid sequence comprises a lysine at position 34, a glutamic acid at position 80, and one or more of:
1) threonine at position 5;
2) a proline at position 16;
3) arginine at position 40;
4) histidine at position 65;
5) a proline at position 92;
6) aspartic acid at position 100;
7) a glutamic acid at position 108; and
8) a threonine at the position 116 of the amino acid sequence,
wherein the specified amino acid residues are located at positions corresponding to SEQ ID NO: 2, the position of the position equivalent; or
iv) comprises a sequence identical to SEQ ID NO: 101, wherein the amino acid sequence comprises a lysine at position 10, a glutamic acid at position 56, and one or more of the following:
1) arginine at position 16;
2) histidine at position 41;
3) a proline at position 68; and
4) (iii) an aspartic acid at position 76,
wherein the specified amino acid residues are located at positions corresponding to SEQ ID NO: 101 at a position equivalent to the position in the first embodiment,
and wherein the polypeptide is capable of hybridizing to a polypeptide comprising SEQ ID NO:5, wherein the isopeptide bond is formed at the amino acid sequence shown in SEQ ID NO:5 and an aspartic acid residue at position 10 of SEQ ID NO: 2 or at position 34 of SEQ ID NO:
101, lysine residue at position 10.
13. A polypeptide according to claim 12, wherein the polypeptide is as defined in any one of claims 4 to 9.
14. A recombinant or synthetic polypeptide comprising a polypeptide and a peptide as defined in claim 10 or 11 and/or a polypeptide as defined in claim 12 or 13.
15. A nucleic acid molecule comprising a nucleotide sequence encoding a peptide as defined in claim 10 or 11, a polypeptide as defined in claim 12 or 13, or a recombinant or synthetic polypeptide according to claim 14.
16. A vector comprising the nucleic acid molecule of claim 15.
17. A cell comprising the nucleic acid of claim 15 or the vector of claim 16.
18. A method of producing or expressing a peptide and/or polypeptide according to any one of claims 10 to 13, comprising the steps of:
a) transforming or transfecting a host cell with a vector comprising a nucleotide sequence encoding a peptide and/or polypeptide as defined in claim 15;
b) culturing said host cell under conditions that allow expression of said peptide and/or polypeptide; and optionally
c) Isolating the peptide and/or polypeptide.
19. Use of a two-part linker peptide according to any one of claims 1 to 9 for conjugating two molecules or components by an isopeptide bond,
wherein said molecule or component conjugated through an isopeptide bond comprises:
a) a first molecule or component comprising the peptide of claim 10 or claim 11; and
b) a second molecule or component comprising the polypeptide of claim 12 or claim 13.
20. A method of conjugating two molecules or components by an isopeptide bond, comprising:
a) providing a first molecule or component comprising the peptide of claim 10 or claim 11;
b) providing a second molecule or component comprising the polypeptide of claim 12 or claim 13;
c) contacting said first and second molecules or components under conditions that allow for spontaneous formation of an isopeptide bond between said peptide and polypeptide, thereby conjugating said first molecule or component to said second molecule or component via an isopeptide bond to form a complex.
21. A kit, preferably for use according to claim 19 or a method according to claim 20, wherein the kit comprises:
(a) the peptide according to claim 10 or claim 11, optionally conjugated or fused to a molecule or component; and
(b) the polypeptide of claim 12 or claim 13, optionally conjugated or fused to a molecule or component; and/or
(c) Nucleic acid molecules, in particular vectors, encoding the peptides defined in (a); and
(d) a nucleic acid molecule, in particular a vector, encoding a polypeptide as defined in (b).
Technical Field
The present invention relates to a two-part linker comprising a peptide tag and a polypeptide (protein) which is capable of spontaneously forming isopeptide bonds. In particular, the two-part linker of the invention may be viewed as a pair of peptide tag and polypeptide binding partner homologues which may be conjugated by covalent bonds when contacted under conditions allowing for the spontaneous formation of isopeptide bonds between the peptide tag and its polypeptide binding partner. Also provided are nucleic acid molecules encoding said each part of the two-part linker (i.e., peptide tag and polypeptide binding partner), vectors comprising the nucleic acid molecules, and host cells comprising the vectors and nucleic acid molecules. Kits comprising the two-part linkers (i.e., peptide tags and polypeptide binding partners) and/or nucleic acid molecules/vectors are also provided. Also provided are methods of producing the two-part linkers (i.e., peptide tags and polypeptide binding partners) and uses of the two-part linkers of the invention.
Background
Cellular function depends on a large number of reversible, non-covalent protein-protein interactions, the precise arrangement of proteins in a complex influencing and determining its function. Thus, the ability to engineer covalent protein-protein interactions may present a new set of opportunities for basic research, synthetic biology, and biotechnology. In particular, conjugation of two or more proteins to form a so-called "fusion protein" can result in molecules having useful properties. For example, clustering together a protein will often greatly enhance biological signals, such as repetitive antigenic structures on a vaccine. Aggrecan proteins with different activities may also produce complexes with improved activity, for example, by substrate channel effects (substrate channel enzymes) of the enzyme.
Typically, covalent protein interactions are mediated through disulfide bonds, but disulfide bonds are reversible, are not suitable for reductive cellular compartments, and can interfere with protein folding. Peptide tags are convenient tools for protein analysis and modification because their small size minimizes interference with protein function. Peptide tags are easy to genetically encode, and their small size can reduce the disruption of interfering with other interactions, the cost of biosynthesis, and the introduction of immunogenicity. However, the interactions between peptide tags and their peptide or polypeptide binding partners rarely have high affinity, which limits their utility in stable complex formation.
Proteins capable of spontaneously forming isopeptide bonds (so-called "isopeptide proteins") have been advantageously used to develop peptide tag/polypeptide binding partner pairs (i.e., two-part linkers) that covalently bind to each other and provide irreversible interaction (see, e.g., WO2011/098772 and WO 2016/193746, both incorporated herein by reference). In this regard, a protein capable of spontaneously forming isopeptide bonds may be expressed as separate fragments to give a peptide tag and a polypeptide binding partner for the peptide tag, wherein the two fragments are capable of covalent reconstitution by isopeptide bond formation to join molecules or components fused to the peptide tag and its polypeptide binding partner. The isopeptide bond formed by the peptide tag and its polypeptide binding partner is stable under conditions in which non-covalent interactions will rapidly dissociate, such as over a long period of time (e.g., weeks), at high temperatures (at least 95 ℃), with high force, or through harsh chemical treatments (e.g., pH 2-11, organic solvents, detergents, or denaturants).
Isopeptide bonds are amide bonds formed between carboxyl/carboxamide groups and amino groups, wherein at least one of the carboxyl or amino groups is outside the protein backbone (the backbone of the protein). Such bonds are chemically irreversible under typical biological conditions, and they are resistant to most proteases. Since isopeptide bonds are covalent in nature, some of the strongest protein interactions can result.
Briefly, a two-part linker, i.e. a peptide tag and its polypeptide binding partner (so-called peptide tag/binding partner pair), may be derived from a protein capable of spontaneously forming isopeptide bonds (isopeptide protein), wherein the domains of the protein are expressed separately to yield a peptide tag comprising one residue associated with an isopeptide bond (e.g. aspartic acid or asparagine) and a peptide or polypeptide binding partner (or "capture agent") comprising another residue involved in the isopeptide bond (e.g. lysine) and at least one other residue required for the formation of an isopeptide bond (e.g. glutamic acid). Mixing the peptide tag and binding partner results in the spontaneous formation of an isopeptide bond between the tag and binding partner. Thus, by fusing the peptide tag and binding partner separately to different molecules or components (e.g., proteins), the molecules or components can be covalently linked together through isopeptide bonds formed between the peptide tag and binding partner, i.e., a linker is formed between the molecules or components fused to the peptide tag and binding partner.
Peptide tag/binding partner pairs (two-part linkers), known as spy tags/spy capture agents (SpyTag/SpyCatcher), are derived from the CnaB2 domain of streptococcus pyogenes FbaB protein (Zakeri et al, 2012, Proc Natl Acad Sci US a 109, E690-697), and are used in a variety of applications, including biomaterials (Botyanszki et al, 2015, Biotechnology and Biotechnology engineering 112, 2016-. However, although the rate of isopeptide bond formation between the spy label and the spy capture agent is satisfactory for purified components, the rate is limited at the level of cellular expression.
Thus, there is a need to develop linkers, such as peptide tag ("tag") and polypeptide binding partner ("capture agent") pairs, that have the advantageous properties associated with tag/capture agent systems derived from isopeptide proteins (i.e., peptide tag and polypeptide binding partner), form stable and strong covalent bonds as described above, with reaction rates high enough to enable efficient reactions at low concentrations, particularly at cellular expression levels.
The inventors have surprisingly determined that the reaction rate of the spy tag and spy capture agent peptides can be significantly increased by modifying (i.e. mutating) the amino acid sequences of the spy tag peptide and spy capture agent polypeptides (seq id NOs: 6 and 7, respectively). As discussed in detail in the examples, a number of steps are required to determine whether the spy label and spy capture agent reaction rates can be increased, and if so, which modifications to the spy label peptide and spy capture agent polypeptide will increase the reaction rates without adversely affecting other desirable properties of the peptide label and binding partner pair.
First, the inventors must determine the extent to which residues in spy tag and spy capture agents, i.e., spy tag peptide (SEQ ID NO: 6) and spy capture agent polypeptide (SEQ ID NO: 7), can be successfully screened for improvement without substantially reducing the reaction rate. It is hypothesized that the activity of the spy tag peptide is largely determined by some "anchor" residues. Since mutations at the anchor residues are likely to mask the effect of mutations at other positions on the reaction rate with only a moderate positive impact, it is postulated that a library of peptide tagged mutants is generated in which any position in the sequence is allowed for mutation, which would actually reduce the likelihood of identifying peptides with improved activity. Thus, the inventors have selected two N-terminal residues of the spy tag and six C-terminal residues of the spy tag for modification and determined that the addition of residues at the N-terminal and/or C-terminal is allowed. A spy capture agent polypeptide library with random mutation is established and used for a screening method. In this regard, it is difficult to design mutations based on the crystal structure of the spy capture agent because not all residues are visible in the crystal structure.
Secondly, the inventor determines that the spy tag mutants at the N end and the C end should be screened separately, and designs a proper screening method to identify the mutant spy tag peptide and the spy capture agent polypeptide with improved activity. Thus, two subsets of the library with mutations at the N-or C-terminus of the spy label were generated and screened for their increased activity in phage display systems using spy capture agents as bait. A separate screen was performed using a spy catcher mutants library (using spy tags as bait).
The design of selection parameters in phage display systems is not straightforward. In this regard, it is hypothesized that the rate at which the spy tag peptide and spy capture agent polypeptide interact may limit the rate of reaction. Therefore, the development of a suitable screening system requires the selection of reaction conditions under which the reaction rate between the spy tag peptide and the spy capture agent polypeptide is not optimal. The use of reaction conditions (e.g. pH, temperature, etc.) where the reaction between the spy label and the spy capture agent is the fastest will prevent the detection of differences in the reactivity of the mutant peptides and polypeptides with respect to the spy label and the spy capture agent, respectively.
Another key to the identification of mutant peptides and polypeptides comes from the design of conditions for separating unreacted mutant tag-capture agent complexes linked by non-covalent bonds from complexes linked by isopeptide bonds. As described in the examples, a combination of low pH buffer and protease treatment was used to separate non-covalent and covalent complexes, thereby ensuring that mutant peptides and polypeptides capable of spontaneously forming isopeptide bonds only with their respective partners were selected for analysis and further modification.
In this regard, the development of mutant "tags" and "capture agents" with improved reaction rates relative to spy tags and spy capture agents requires the design and introduction of various other modifications (i.e., mutations) to mutant peptides and polypeptides identified from the screening process. The modifications not only result in mutant "tag" and "capture agent" polypeptides having an increased reaction rate when reacted with their unmutated partners (e.g., > 6 fold increase in mutant tags reacted with unmutated spy capture agents and > 3 fold increase in mutant capture agents reacted with unmutated spy tags), but surprisingly it was determined that the effect of mutations on the reaction rate of mutant "tags" and "capture agents" is cumulative when used together (i.e., > 10 fold increase in reaction rate relative to the spy tag and spy capture agent pair). Thus, advantageously, the mutant tags and capture agents (i.e., two-part linkers) of the present invention are particularly useful at low concentrations. As discussed further below, the improved rate constants of the mutant tags and captures of the invention are also advantageous in reactions in which the tag and/or capture agent is fused to a molecule or component (e.g., a large protein) that may slow the reaction, as well as in reactions in which the molecule or component fused to the mutant tag and/or capture of the invention causes steric hindrance. Moreover, the modifications required to increase the speed of the reaction do not affect other useful properties associated with spy-tags and spy-traps, i.e. thermostability, reaction across a range of pH and temperature, and in a wide range of buffers including the presence of detergents and high expression in e.
Disclosure of Invention
Thus, in one aspect, the present invention therefore provides a peptide, i.e. a peptide tag, comprising a sequence as set forth in SEQ ID NO:1, wherein:
(i) x at
(ii) x at
(iii) x at
(iv) x at position 11 is alanine, glycine or valine, preferably alanine; and
(v) x at position 14 is arginine or lysine, preferably arginine,
wherein when X at
And wherein the peptide (peptide tag) is capable of hybridizing to a peptide comprising SEQ ID NO: 2 (i.e. a polypeptide binding partner) spontaneously forms an isopeptide bond, wherein the isopeptide bond is formed at the amino acid sequence shown in SEQ ID NO:1 and the aspartic acid residue at
Thus, the peptide tags of the present invention comprise at least four (e.g. five or six) modifications (e.g. additions and substitutions) relative to the original spy tag peptide.
As described in the examples below, the lead mutant peptide tag (spy tag variant peptide) identified in the N-terminal screen contained 3N-terminal amino acids relative to the spy tag and was determined to remove two of the residues without significantly affecting the reaction rate of the peptide. Thus, in some embodiments, the peptide tag of the invention is set forth in SEQ ID NO:1, i.e. when X at
(i) x at
(ii) x at position 9 is alanine, glycine or valine, preferably alanine; and
(iii) x at position 12 is arginine or lysine, preferably arginine.
However, the inventors have determined that the inclusion of arginine and glycine residues at the N-terminus further increases the reaction rate of spy tag variants. Thus, in a preferred embodiment, the peptide tag of the invention comprises SEQ ID NO:1, wherein:
(i) x at
(ii) x at
(iii) x at
(iv) x at position 11 is alanine, glycine or valine, preferably alanine; and
(v) x at position 14 is arginine or lysine, preferably arginine,
in another aspect, in some embodiments, the peptide tag comprises SEQ ID NO:9 or consisting of the amino acid sequence set forth in seq id no:
(i) x at
(ii) x at position 11 is alanine, glycine or valine, preferably alanine; and
(iii) x at position 14 is arginine or lysine, preferably arginine.
It is contemplated that the sequences set forth in SEQ ID NO:1 and 9 (equivalent to positions 9 and 12 of SEQ ID NO: 8) without significantly affecting the activity of the peptide tag. However, in some embodiments, it is preferred that SEQ id no:1 and 9 (equivalent to position 9 of SEQ ID NO: 8) is alanine and/or SEQ ID NO: position 14 of 1 and 9 (equivalent to position 12 of SEQ ID NO: 8) is an arginine.
The lead mutant peptide tag (spy tag variant peptide) identified in the N-terminal screen described in the examples contained a valine residue at
Thus, in a preferred embodiment, the peptide tag of the invention comprises SEQ ID NO:1, wherein:
(i) x at
(ii) x at
(iii) x at
(iv) x at position 11 is alanine, glycine or valine, preferably alanine; and
(v) x at position 14 is arginine or lysine, preferably arginine,
in another aspect, in some embodiments, the peptide tag of the invention comprises SEQ ID NO:10, wherein:
(i) x at position 11 is alanine, glycine or valine, preferably alanine;
(ii) x at position 14 is arginine or lysine, preferably arginine.
Thus, in some embodiments, the peptide tag of the invention comprises SEQ ID NO: 3. 4 or 5, preferably SEQ ID NO:4 or 5, most preferably SEQ ID NO: 5.
As described above, phage display screens have identified variant (i.e., mutant) polypeptides (peptide tag binding partners or capture agents) with improved activity relative to spy capture agents. It is contemplated that each substitution in the polypeptide (peptide tag binding partner) of the present invention (SEQ ID NO: 2, spy capture agent polypeptide variant) may increase the activity of the polypeptide (peptide tag binding partner) with respect to the spy capture agent amino acid sequence (SEQ ID NO: 7), respectively.
Furthermore, in view of the fact that spy catcher polypeptides may be truncated at their N-and C-termini without significantly affecting their activity (Li et al, 2014, J Mol biol.; 426(2):309 and 317), it is contemplated that the polypeptides exemplified herein (i.e., SEQ ID NO: 2) may be truncated at the N-and/or C-termini without significantly reducing the activity of the polypeptide. In particular, SEQ ID NO: 2 may be truncated by up to 24 amino acids (e.g., 5, 10, 15, or 20 amino acids) at the N-terminus and/or up to 9 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7,8, or 9 amino acids) at the C-terminus.
Thus, in another aspect, the present invention provides a polypeptide (peptide tag binding partner) comprising:
i) SEQ ID NO: 2;
ii) comprises SEQ ID NO: 101, or (ii) a portion of (i) the amino acid sequence set forth in seq id no;
iii) and SEQ ID NO: 2, wherein the amino acid sequence comprises a lysine at
1) threonine at
2) a proline at position 16;
3) arginine at
4) histidine at position 65;
5) a proline at position 92;
6) aspartic acid at
7) a glutamic acid at position 108; and
8) a threonine at the position 116 of the amino acid sequence,
wherein the specified amino acid residues are located at positions corresponding to SEQ ID NO: 2, the positions in 2 are equivalent; or
iv) comprises a sequence identical to SEQ ID NO: 101 (iii) having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 101), wherein the amino acid sequence comprises a lysine at position 10 (or a position equivalent to
1) at position 16 (or equivalent to SEQ ID NO:
2) at position 41 (or equivalent to SEQ ID NO: position 65 in 2);
3) at position 68 (or equivalent to SEQ ID NO: 2 at position 92) proline;
4) at position 76 (or equivalent to SEQ ID NO:
wherein the specified amino acid residues are located at positions corresponding to SEQ ID NO: 101 (or SEQ ID NO: 2),
and wherein the polypeptide is capable of hybridizing to a polypeptide comprising SEQ ID NO:5 (peptide tag) spontaneously forms an isopeptide bond, wherein the isopeptide bond is formed in SEQ ID NO:5 and an aspartic acid residue at
In embodiments of the invention wherein the polypeptide (peptide tag binding partner) variant (i.e. the polypeptide related to sequence identity and parts thereof) does not comprise all of the residues specified above, it is preferred that the variant comprises the amino acid residue at the equivalent position of the spy capture agent peptide (SEQ ID NO: 7) at the specified position, with the exception of position 5 (discussed below). By comparing the amino acid sequence of the polypeptide (peptide tag binding partner) variant with the amino acid sequence of SEQ ID NO: equivalent positions can be readily determined, for example, using the BLASTP algorithm.
Thus, for example, where a polypeptide (peptide tag binding partner) of the invention comprises a sequence identical to SEQ ID NO: 2, if the residue at position 16 (or equivalent position) is not proline, it is preferred that the residue is glutamine. Similarly, if the residue at position 40 (or equivalent position) is not arginine, then preferably the residue is lysine. If the residue at position 65 (or equivalent position) is not histidine, it is preferred that the residue is glutamine. If the residue at position 92 (or equivalent position) is not proline, it is preferred that the residue is alanine. If the residue at position 100 (or equivalent position) is not aspartic acid, it is preferred that the residue is glutamine. If the residue at position 108 (or equivalent position) is not glutamic acid, it is preferred that the residue is lysine. If the residue at position 116 (or equivalent position) is not threonine, it is preferred that the residue is isoleucine.
In some embodiments, a polypeptide (peptide tag binding partner) variant of the present invention may be compared to SEQ ID NO: 2 phase differences, for example 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 8,1 to 6,1 to 5, 1 to 4, for example 1, 2 or 3 amino acid substitutions, insertions and/or deletions, preferably 1 to 23, 1 to 20, 1 to 15, 1 to 10, 1 to 8,1 to 6,1 to 5, 1 to 4, for example 1, 2 to 3 amino acid substitutions and/or 1 to 33, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 8,1 to 6,1 to 5, 1 to 4, for example 1, 2 or 3 amino acid deletions. As described below, in some embodiments, it is preferred that the deletion is at the N-and/or C-terminus, i.e. truncated, thereby generating the amino acid sequence of SEQ ID NO: 2.
In some embodiments, any mutation present in a polypeptide (peptide tag binding partner) of the present invention relative to an exemplary polypeptide (SEQ ID NO: 2) may be a conservative amino acid substitution. A conservative amino acid substitution is one in which the amino acid is substituted with another amino acid while retaining the physicochemical properties of the polypeptide (e.g., D may be replaced with E, and vice versa, N may be replaced with Q, L or I may be replaced with V, and vice versa). Thus, in general, a substituted amino acid has similar properties as a substituted amino acid, such as hydrophobicity, hydrophilicity, electronegativity, side chain volume, and the like. Isomers of natural L-amino acids, such as D-amino acids, may be incorporated.
Thus, in some embodiments in which a polypeptide (peptide tag binding partner) variant of the present invention does not comprise all of the residues specified above (i.e., all mutations in SEQ ID NO: 2 relative to SEQ ID NO: 7), the variant may comprise conservative substitutions of amino acid residues at equivalent positions of the spy capture agent peptide (SEQ ID NO: 7) at specified positions, other than
Thus, in some embodiments, a polypeptide (peptide tag binding partner) of the invention may comprise a sequence identical to SEQ ID NO: 2, wherein the amino acid sequence comprises a lysine at
1) threonine at
2) a proline at position 16;
3) arginine at
4) histidine at position 65;
5) a proline at position 92;
6) aspartic acid at
7) a glutamic acid at position 108; and
8) a threonine at the position 116 of the amino acid sequence,
wherein the specified amino acid residues are located at positions corresponding to SEQ ID NO: 2, are equivalent positions.
As described in the examples below, the inventors have unexpectedly determined the presence of an aspartic acid residue at position 5 (based on numbering of SEQ ID NO: 2 and SEQ ID NO: 7) of a mutant (i.e., variant) of a polypeptide (peptide tag binding partner) that has been identified in phage display screens as resulting in the formation of an undesirable side reaction-polypeptide (peptide tag binding partner) dimer in which the polypeptides are conjugated via isopeptide bonds. It was shown that mutation of the aspartate residue at
1) a proline at position 16;
2) arginine at
3) histidine at position 65;
4) a proline at position 92;
5) aspartic acid at
6) a glutamic acid at position 108; and
7) a threonine at the position 116 of the amino acid sequence,
wherein the specified amino acid residues are located at positions corresponding to SEQ ID NO: 2, are equivalent positions.
It is contemplated that the polypeptide (peptide tag binding partner) of the invention may comprise any one or any combination of the specified amino acid residues defined above (e.g., any combination of two, three, four, five, six, or seven of the above specified amino acid residues), e.g., 1) and 2), 1) and 3), 1 and 4), 1) and 5), 1) and 6), 1) and 7), 1) and 8), 2) and 3), 2) and 4), etc., 1), 2) and 3), 1), 3) and 4), 1), 3) and 5), etc. However, some particularly preferred combinations include:
a)1) threonine at
2) a proline at position 16;
3) a lysine at
4) arginine at
5) histidine at position 65;
6) glutamic acid at
7) A glutamic acid at position 108; and
8) threonine at position 116;
b)1) threonine at
2) a proline at position 16;
3) a lysine at
4) arginine at
5) histidine at position 65;
6) glutamic acid at
7) Proline at position 92
8) A glutamic acid at position 108; and
9) threonine at position 116; and
c)1) threonine at
2) a proline at position 16;
3) a lysine at
4) arginine at
5) histidine at position 65;
6) glutamic acid at
7) Proline at position 92
8) Aspartic acid at
9) A glutamic acid at position 108; and
10) a threonine at the position 116 of the amino acid sequence,
wherein the specified amino acid residues are located at positions corresponding to SEQ ID NO: 2, are equivalent positions.
In some other embodiments, the polypeptide (peptide tag binding partner) variant as defined above may further comprise a glycine at position 12 and/or a threonine at position 22.
Thus, the polypeptides (peptide tag binding partners) of the invention may in particular be linked to SEQ ID NO: 2, and more particularly to SEQ ID NO: 2 at least 85, 90, 95, 96, 97, 98, 99 or 99% identical, wherein the polypeptide variant comprises a lysine at position 34 (or equivalent position), a glutamic acid at position 80 (or equivalent position), and one or more of:
1) threonine at
2) a proline at position 16;
3) arginine at
4) histidine at position 65;
5) a proline at position 92;
6) aspartic acid at
7) a glutamic acid at position 108; and
8) a threonine at the position 116 of the amino acid sequence,
wherein the specified amino acid residues are located at positions corresponding to SEQ ID NO: 2, are equivalent positions.
The term "linker" as used herein refers to a molecule that functions to link (i.e., conjugate or join) two molecules or components, preferably by covalent bonding, for example by isopeptide bonding. Thus, the peptide tag and polypeptide of the present invention may be viewed as a two-part linker, wherein the formation of an isopeptide bond between a first part, i.e., the peptide tag, and a second part, i.e., the polypeptide, reconfigures the linker, thereby linking the molecules or components fused or conjugated to the first and second parts of the linker. In other words, the peptide tag and polypeptide of the present invention may be considered as a pair of homologues having a linker function, i.e. a pair of peptide tag and polypeptide homologues or a pair of peptide tag and binding partner homologues. These terms are used interchangeably throughout the specification.
The term "homologue" refers to components that work together. Thus, in the context of the present invention, a pair of homologues refers to the peptide tag and the polypeptide of the present invention, which react spontaneously together to form an isopeptide bond. Thus, a two-part linker comprising a peptide tag and a polypeptide, which effectively react together to form an isopeptide bond under conditions capable of spontaneously forming said isopeptide bond, may also be referred to as a "complementary pair", i.e. a complementary pair of peptide tag and polypeptide.
Thus, the present invention further provides a two-part linker comprising a peptide (peptide tag) and a polypeptide (peptide tag binding partner), wherein:
a) the peptide (peptide tag) comprises an amino acid sequence as defined above; and
b) the polypeptide (peptide tag binding partner) comprises an amino acid sequence as defined above,
wherein the peptide (peptide tag) and polypeptide (peptide tag binding partner) are capable of spontaneously binding to the polypeptide of SEQ ID NO:1 and an aspartic acid residue at
The peptide tags and polypeptides (peptide tag binding partners) of the invention spontaneously occur in SEQ ID NO:1 and the aspartic acid residue at
For example, peptide tags and polypeptides (peptide tag binding partners) are active in a variety of buffers, including Phosphate Buffered Saline (PBS), 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), HEPES Buffered Saline (HBS), Tris Buffered Saline (TBS), with or without EDTA. The peptide tags and polypeptides (peptide tag binding partners) are active at a pH of about 3.0-8.0 (e.g.4.0-7.0, 5.0-7.0, e.g.about 5.5-6.5) over a wide temperature range, e.g.0-40 deg.C, e.g.1, 2, 3, 4, 5, 10, 12, 15, 18, 20, 22, 25, 28, 30, 35 or 37 deg.C, preferably about 25-35 deg.C, e.g.about 25 deg.C. The peptide tags and polypeptides (peptide tag binding partners) of the invention are also active in the presence of commonly used detergents such as
Thus, in some embodiments, conditions suitable for the formation of an isopeptide bond between the peptide tag of the invention and a polypeptide (peptide tag binding partner) include any condition that brings the peptide tag and the polypeptide (peptide tag binding partner) into contact. The present invention results in a polypeptide (peptide tag binding partner) that binds specifically between the peptide tag and the polypeptide, in particular between SEQ ID NO:1 (or an equivalent position) with an aspartic acid residue at
In some embodiments, contacting a peptide tag of the invention with a polypeptide (peptide tag binding partner) "under conditions capable of spontaneously forming isopeptide bonds" comprises contacting the peptide tag with the polypeptide in the presence of a chemical chaperone. Such as molecules that enhance or improve the reactivity of peptide tags and polypeptides (peptide tag binding partners). In some embodiments, the chemical chaperone is TMAO (trimethylamine N-oxide). In some embodiments, a chemical chaperone, such as TMAO, is present in the reaction at a concentration of at least about 0.2M, for example at least 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, or 2.5M, for example about 0.2-3.0M, 0.5-2.0M, 1.0-1.5M.
As mentioned above, the formation of an isopeptide bond between the peptide tag of the invention and a polypeptide (peptide tag binding partner) is spontaneous. In this regard, the polypeptide (peptide tag binding partner) comprises a glutamic acid at position 80 (or equivalent position based on the numbering of SEQ ID NO: 2) which is useful, for example, to induce, promote or catalyze the formation of isopeptide bonds between aspartic acid and lysine residues in the peptide tag and the polypeptide (peptide tag binding partner), respectively.
As used herein, the term "spontaneous" refers to isopeptide bonds that can be formed in or between peptides or proteins (e.g., between two peptides or between a peptide and a protein, i.e., between a peptide tag and a polypeptide of the invention (peptide tag binding partner)) without the presence of any other agent (e.g., an enzyme catalyst) and/or without chemical modification of the protein or peptide, e.g., without the use of native chemical ligation or chemical coupling using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). Thus, native chemical ligation was not performed to modify a peptide or protein with a C-terminal thioester.
Thus, when isolated and without chemical modification of the peptide tags and/or polypeptides of the invention, spontaneous isopeptide bonds may form between the peptide tags of the invention and the polypeptides (peptide tag binding partners). Thus, spontaneous isopeptide bonds can form themselves in the absence of enzymes or other exogenous substances and without chemical modification of the peptide tags and/or polypeptides of the invention.
Spontaneous isopeptide bonds can form almost immediately upon contact of the peptide tag of the invention with a polypeptide (peptide tag binding partner), for example within 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30 minutes, or within 1, 2, 4, 8, 12, 16, 20 or 24 hours.
The peptide tags and polypeptides (peptide tag binding partners) of the present invention include mutated forms (i.e., referred to herein as homologs, variants or derivatives) of peptide tags and polypeptides (peptide tag binding partners) that are structurally identical to the exemplified peptide sequences of SEQ id nos: 3-5 and the peptide tag shown in SEQ ID NO: 2 (peptide tag binding partner) are similar. The peptide tag and polypeptide (peptide tag binding partner) variants of the invention are capable of acting as a peptide tag and binding partner (capture agent), i.e. capable of spontaneously forming an isopeptide bond between an aspartic acid at position 10 (or equivalent position) of the peptide tag variant and a lysine at position 34 (or equivalent position) of the polypeptide (peptide tag binding partner) variant under suitable conditions as defined above.
In the case of peptide tags or polypeptide (peptide tag binding partner) variants, respectively, relative to SEQ ID NO:1 and 2 comprise a mutation (e.g., a deletion or an insertion), the residues specified above are present at equivalent amino acid positions in the sequence of the variant peptide tag and the polypeptide (peptide tag binding partner). In some embodiments, the deletions in the peptide tags and polypeptide (peptide tag binding partner) variants of the invention are not N-terminal and/or C-terminal truncations.
However, as noted above, it is contemplated that the polypeptides exemplified herein (i.e., SEQ ID NO: 2) can be truncated at the N-terminus and/or C-terminus without significantly reducing the activity of the polypeptide. In particular, SEQ ID NO: 2 may be truncated at the N-terminus by up to 24 amino acids (e.g., 5, 10, 15, or 20 amino acids) and/or at the C-terminus by up to 9 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7,8, or 9 amino acids). Thus, the term variant as used herein includes truncated variants of the exemplary polypeptides. Viewed from a further aspect, it can be seen that the present invention provides a portion of an exemplary polypeptide, wherein said portion comprises the amino acid sequence of SEQ ID NO: 101, or a variant thereof.
As referred to herein, a "portion" (part) comprises at least SEQ ID NO: 101, i.e. comprising the amino acid sequence shown in SEQ ID NO: 101, at least 83, 84, 85, 86, 87, 88, 89, 90, 95, 100, 105, 110 or more of the amino acid sequences set forth in SEQ ID NOs: 2 (sequence from which it is derived). Thus, the portion may be obtained from the central or N-terminal or C-terminal portion of the sequence. Preferably, the moiety is obtained from the central part, i.e. it comprises an N-terminal and/or C-terminal truncation as defined above. In particular, a "portion" as described herein is a polypeptide of the invention, and thus satisfies the identity (relative to comparable regions) conditions and functional equivalents mentioned herein.
In some embodiments, the peptide tag variants of the invention may be compared to SEQ ID NO:1, e.g. 1 to 5, 1 to 4, e.g. 1, 2 to 3 amino acid substitutions, insertions and/or deletions as described above, preferably substitutions. In some embodiments, the polypeptide (peptide tag binding partner) variants of the invention may differ from the amino acid sequence of SEQ ID NO: 2.
sequence identity may be determined by any suitable method known in the art. The SWISS-PROT protein sequence database was used, FASTA pep-cmp with variable pam factor was used, and gap creation penalty was set to 12.0 and gap extension penalty was set to 4.0, and contained a window of 2 amino acids. Other programs for determining amino acid sequence identity include the BestFit program of the Genetic Computer Group (GCG)
Preferably, the comparison is made over the entire length of the sequence, but may be made over a smaller comparison window, e.g., less than 100, 80, or 50 contiguous amino acids.
Preferably, the peptide tag and polypeptide (peptide tag binding partner) variants (e.g., sequence identity-related variants) are functionally equivalent to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 3-5 or SEQ ID NO: 2 or 101 and a polypeptide (peptide tag binding partner). As referred to herein, "functionally equivalent" refers to variants of the peptide tags and polypeptides (peptide tag binding partners) of the invention discussed above that exhibit reduced efficacy (e.g., lower expression yield relative to the parent molecule (i.e., the molecule with which they exhibit sequence homology), lower reaction rate, or activity over a limited range of reaction conditions (e.g., a narrower temperature range, such as 10-30 ℃, etc.) upon spontaneous formation of isopeptide bonds with their respective partners, but preferably are equally or more effective.
Has a sequence similar to that of a sequence comprising SEQ ID NO: 3-5 or a peptide tag consisting thereof, may have an activity "equivalent" to the activity of a peptide tag comprising or consisting of SEQ ID NO: 3-5 or a peptide tag consisting thereof, i.e. such that the actual application of the peptide tag is not significantly affected, e.g. within experimental error. Thus, equivalent peptide tag activity means that a mutant or variant peptide tag of the invention is capable of reacting with a polypeptide (peptide tag binding partner, e.g., comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2, 7, or 101) at a similar reaction rate (i.e., the rate constant as described below) and/or yield to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3-5 under the same conditions spontaneously form an isopeptide bond.
Similarly, a polypeptide comprising SEQ ID NO: 2 or 101 (preferably SEQ ID NO: 2) or a polypeptide consisting thereof (peptide tag binding partner) may have an activity which is "equivalent" to the activity of a mutant or variant polypeptide (peptide tag binding partner) of the invention comprising an amino acid sequence as set forth in SEQ ID NO: 2 or 101 (preferably SEQ ID NO: 2) or a polypeptide (peptide tag binding partner) consisting thereof, i.e. such that the actual use of the polypeptide (peptide tag binding partner) is not significantly affected, e.g. within experimental error. Thus, equivalent polypeptide (peptide tag binding partner) activity means that a mutant or variant polypeptide (peptide tag binding partner) of the invention is capable of reacting with a peptide tag (e.g., comprising or consisting of an amino acid sequence as set forth in one of SEQ ID NOs: 3-6) at a similar reaction rate (i.e., a rate constant as described below) and/or yield to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NOs: 2 or 101 (preferably SEQ ID NO: 2) under the same conditions spontaneously form isopeptide bonds.
The activity of different peptide tags and polypeptides (e.g., SEQ ID NO:5 versus mutants, or SEQ ID NO: 2 versus mutants) under the same reaction conditions can be readily compared at the temperatures described above, the substrate (i.e., peptide tag or polypeptide sequence) and its concentration, buffer, salt, etc., to determine whether the activity of each peptide tag and polypeptide is higher, lower, or equivalent.
In particular, the peptide tags and polypeptide variants of the invention are compared to polypeptides having the amino acid sequences as set forth in SEQ ID NOs: 3-5 or SEQ ID NO: 2 or 101 has an equivalent rate constant. The rate constant refers to the proportionality coefficient of the reaction (isopeptide bond formation) rate multiplied by the reactant concentration (i.e., the product of the concentration of the peptide tag and polypeptide of the invention) at a given temperature.
Thus, the activity, e.g., rate constant, of the variant (e.g., mutant) peptide tag can be a peptide comprising or consisting of SEQ id no: 3-5, such as at least 60%, such as at least 70, 75, 80, 85 or 90% of the rate constant, as determined for a peptide tag comprising the amino acid sequence set forth in one of SEQ ID NOs: 3-5 or a peptide tag consisting thereof, at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% of the activity of the peptide tag. Alternatively, the activity of the mutant peptide tag, e.g., the rate constant, can be greater than the activity of a polypeptide comprising or consisting of SEQ ID NO: 3-5, e.g. the rate constant, is not more than 40% lower than the activity of a peptide tag comprising or consisting of the amino acid sequence as set forth in any one of SEQ ID NOs: 3-5, e.g. a rate constant, which is not more than 35%, 30%, 25% or 20% lower, such as compared to a peptide tag comprising or consisting of the amino acid sequence as set forth in any one of SEQ ID NOs: 3-5, e.g., the rate constant, is not less than 10, 9, 8, 7, 6, 5, 4,3, 2, or 1%.
Similarly, the activity, e.g., rate constant, of a variant (e.g., mutant) polypeptide (peptide tag binding partner) of the invention can be a polypeptide comprising or consisting of SEQ ID NO: 2 or 101, such as at least 60%, such as at least 70, 75, 80, 85 or 90% of the rate constant, as determined for a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or 101 or a polypeptide consisting thereof, e.g., at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% of the rate constant. In another aspect, the activity of the mutant polypeptide can be greater than the activity of a polypeptide comprising SEQ ID NO: 2 or 101 or a polypeptide consisting thereof, e.g. a rate constant, is not less than 40%. For example, compared to a polypeptide comprising SEQ ID NO: 2 or 101 or a polypeptide consisting thereof, e.g., a rate constant, that is NO more than 35%, 30%, 25% or 20% lower, as compared to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 101 or a polypeptide consisting thereof, e.g., a rate constant, is no less than 10, 9, 8, 7, 6, 5, 4,3, 2, or 1%.
It is noteworthy that the rate constant of the reaction of the peptide tag and polypeptide of the invention may be lower than the values described in the examples when the peptide tag and/or polypeptide is fused to a macromolecule or component (e.g., a protein) that diffuses more slowly than the isolated peptide tag and polypeptide. Furthermore, the rate constant may be reduced if the molecule or component to which the peptide tag and/or polypeptide is fused sterically hinders the reaction. Thus, when measuring the rate constants of the reactions of the peptide tags and polypeptide variants of the invention, preferably the measurements are performed using isolated peptide tags and polypeptides, i.e. peptide tags and polypeptides that are not fused or conjugated to other molecules or components.
Clearly, fusion and/or steric hindrance with macromolecules or components will also affect the rate constants of other peptide tags and polypeptides, such as spy tags and spy capture agents. Thus, in addition to being applied at low concentrations, the enhancement of the rate constants of the peptide tags and polypeptides of the present invention may still be advantageous when the peptide tags and polypeptides of the present invention are used at high concentrations (e.g., when fused to macromolecules or components).
The rate of reaction and rate constants can be assessed by any suitable method known in the art and as described in the examples. For example, the reaction rate can be monitored by assessing the mobility of reaction products on SDS-PAGE after boiling in SDS or other strongly denaturing treatments that disrupt all non-covalent interactions or by mass spectrometry.
Thus, the sequence of SEQ ID NO: 2 to produce a variant polypeptide (peptide tag binding partner) of the invention, provided that the variant polypeptide (peptide tag binding partner) is comprised in a sequence equivalent to SEQ ID NO: 2, and a lysine residue at
In another aspect, the sequence of SEQ ID NO: 101 to produce a variant polypeptide (peptide tag binding partner) of the invention, provided that the variant polypeptide (peptide tag binding partner) is comprised in a sequence equivalent to SEQ ID NO: 101, comprises a lysine residue at
Equivalent positions in the peptide tag of the invention are preferably determined by reference to SEQ ID NO:1 or 5. Equivalent positions in the polypeptides (peptide tag binding partners) of the invention are identified by reference to SEQ ID NO: 2 or 101. By aligning the sequence of the homologous (mutant, variant or derivative) peptide tag with the sequence of SEQ ID NO:1 or 5 or a homologous (mutant, variant or derivative) polypeptide (peptide tag binding partner) and the sequence of SEQ ID NO: 2 or 101, homology or corresponding positions can be easily deduced based on homology or identity between the sequences. For example using the BLAST algorithm.
The terms "tag" and "peptide tag" as used herein generally refer to a peptide or oligopeptide.
As used herein, the term "peptide tag binding partner", "binding partner" or "capture agent" generally refers to a polypeptide or protein.
In this respect, there is no standard definition of the size boundary between peptides or oligopeptides. Generally, peptides can be considered to comprise 2-20 amino acids and oligopeptides comprise 21-39 amino acids. Thus, a polypeptide may be considered to comprise at least 40 amino acids, preferably at least 50, 60, 70, 80, 90, 100 or 110 amino acids.
Thus, in preferred embodiments, a peptide tag as defined herein may be regarded as comprising at least 12 amino acids, such as 12-39 amino acids, for example 13-35, 14-34, 15-33, 16-31, 17-30 amino acids in length, for example may comprise or consist of 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.
A polypeptide of the invention (peptide tag binding partner, binding partner or "capture agent") as defined herein may be regarded as comprising at least 80 amino acids, such as 80-150 amino acids, for example 80-140, 80-130, 80-120 amino acids in length, e.g. it may comprise or consist of 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120 amino acids.
As noted above, two-part linkers (e.g., a tag and capture agent system or pair, i.e., a homolog pair) have a number of utilities, and the peptide tags and polypeptides (peptide tag binding partners) of the present invention find particular utility in conjugating (i.e., linking or joining) the isopeptide bonds of two molecules or components. For example, a peptide tag and a polypeptide (peptide tag binding partner) may be coupled or fused to a target molecule or component, respectively, and then contacted together under conditions suitable for the spontaneous formation of an isopeptide bond between the peptide tag and the polypeptide (peptide tag binding partner), thereby joining (i.e., linking or conjugating) the molecules or components via isopeptide bonds.
Thus, in some embodiments, it may be seen that the present invention provides the use of a pair of peptides (peptide tags) and polypeptides (peptide tag binding partners) as defined herein for conjugating two molecules or components by isopeptide bond, wherein the compounds to which the molecules or components are conjugated by isopeptide bond include:
a) a first molecule or component comprising (e.g., conjugated or fused to) a peptide (peptide tag) of the invention; and
b) a second molecule or component comprising (e.g. conjugated to or fused to) a polypeptide (peptide tag binding partner) of the invention.
It will be apparent that the use of a peptide tag and polypeptide (peptide tag binding partner) pair (i.e. a two-part linker) as described above comprises contacting the first and second molecules under conditions suitable to cause (e.g. promote or facilitate) the spontaneous formation of an isopeptide bond between the peptide tag and polypeptide (peptide tag binding partner) as described above.
In another aspect, the present invention provides a method of binding two molecules or components by isopeptide bond, the method comprising:
a) providing a first molecule or component comprising (e.g. conjugated to or fused to) a peptide (peptide tag) of the invention;
b) providing a second molecule or component comprising (e.g. conjugated to or fused to) a polypeptide (peptide tag binding partner) of the invention;
c) contacting the first and second molecules or components under conditions that cause (e.g., promote or promote) the spontaneous formation of an isopeptide bond between the peptide and polypeptide as described above, thereby conjugating the first molecule or component to the second molecule or component via an isopeptide bond to form a complex.
In the context of the present invention, the term "conjugation" or "linking" when referring to the joining of two or more molecules or components to form a complex means that the molecules or components, e.g. proteins, are bound or conjugated by means of covalent bonds, in particular isopeptide bonds formed between and bound to or fused to the peptide tag and polypeptide (peptide tag binding partner), e.g. proteins (e.g. peptide tag and polypeptide (peptide tag binding partner) may form domains of the proteins to be conjugated or linked together).
As noted above, in some embodiments, the peptide tags and/or polypeptides (peptide tag binding partners) of the present invention are fused or conjugated to other molecules or other components or entities. Such molecules or components (i.e., entities) may be nucleic acid molecules, proteins, peptides, small molecule organic compounds, fluorophores, metal-ligand complexes, polysaccharides, nanoparticles, nanotubes, polymers, cells, viruses, virus-like particles, or any combination thereof. In some embodiments, the component or entity fused or conjugated to the peptide tag and/or polypeptide (peptide tag binding partner) is a solid support, i.e. a solid substrate or solid phase as defined below.
Thus, in another aspect, the invention provides a solid support comprising a nucleic acid molecule, protein, peptide, small molecule organic compound, fluorophore, metal-ligand complex, polysaccharide, nanoparticle, nanotube, polymer, cell, virus-like particle, or any combination thereof fused or conjugated to a peptide tag and/or polypeptide (peptide tag binding partner) of the invention.
The cell may be prokaryotic or eukaryotic. In some embodiments, the cell is a prokaryotic cell, such as a bacterial cell.
In some embodiments, the peptide tag and/or polypeptide (peptide tag binding partner) may be conjugated or fused to a compound or molecule having a therapeutic or prophylactic effect, such as an antibiotic, an antiviral, a vaccine, an antineoplastic agent, e.g., a radioactive compound or isotope, a cytokine, a toxin, an oligonucleotide, and a nucleic acid encoding a gene or nucleic acid vaccine.
In some embodiments, the peptide tag and/or polypeptide (peptide tag binding partner) may be conjugated or fused to a label, such as a radiolabel, a fluorescent label, a luminescent label, a chromophore label, and an enzyme that generates a detectable substrate, such as horseradish peroxidase, luciferase, or alkaline phosphatase. This assay can be used in a variety of assays that routinely use antibodies, including forms of western blot/immunoblot, histochemistry, enzyme-linked immunosorbent assay (ELISA), or flow cytometry (FACS). Labels for magnetic resonance imaging, positron emission tomography probes, and
In particularly useful embodiments, the peptide tag and/or polypeptide (peptide tag binding partner) is fused or conjugated to another peptide, oligopeptide or polypeptide. For example, a peptide tag and/or polypeptide (peptide tag binding partner) may be produced as part of another peptide, oligopeptide or polypeptide, i.e., a recombinant or synthetic protein or polypeptide, using recombinant techniques discussed below.
It will be apparent that the peptide tags and/or polypeptides (peptide tag binding partners) of the invention may be fused to any protein or polypeptide. The protein may be derived or obtained from any suitable source. For example, the protein may be translated or purified from biological and clinical samples, such as any cell or tissue sample of an organism (eukaryotic, prokaryotic), or any body fluid or preparation derived therefrom, as well as samples such as cell cultures, cell preparations, cell lysates and the like. The protein may be derived or obtained, for example purified from environmental samples (e.g. also including soil and water-like or food-like). The sample may be freshly prepared or may be pre-treated in any convenient manner, for example for storage.
As mentioned above, in a preferred embodiment, the peptide, oligopeptide or protein fused to and/or a polypeptide (peptide tag binding partner) may be produced recombinantly, and thus the nucleic acid molecule encoding the recombinant protein may be derived from or obtained from any suitable source, e.g. any viral or cellular material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasma, protoplasts and organelles. Thus, such biological materials may comprise all types of mammalian and non-mammalian animal cells, plant cells, algae (including blue-green algae), fungi, bacteria, protozoa, viruses, and the like. In some embodiments, the protein may be a synthetic protein. For example, the peptides and polypeptides (proteins) disclosed herein can be produced by chemical synthesis, e.g., solid phase peptide synthesis.
The position of the peptide tag and/or polypeptide (peptide tag binding partner) in the recombinant or synthetic protein is not particularly important. Thus, in some embodiments, the peptide tag and/or polypeptide (peptide tag binding partner) may be located at the N-terminus or C-terminus of the recombinant or synthetic polypeptide. In some embodiments, the peptide tag and/or polypeptide (peptide tag binding partner) may be internal to a recombinant or synthetic polypeptide. Thus, in some embodiments, the peptide tag and/or polypeptide (peptide tag binding partner) may be considered an N-terminal, C-terminal, or internal domain of a recombinant or synthetic polypeptide.
In some preferred embodiments, the polypeptide (peptide tag binding partner) is preferably located at the N-terminus or C-terminus of the recombinant or synthetic polypeptide. Thus, in some embodiments, a polypeptide (peptide tag binding partner) may be considered an N-terminal, or C-terminal domain of a recombinant or synthetic polypeptide.
In some embodiments, it is useful to include one or more spacers (e.g. peptide spacers) between the peptides, oligopeptides or polypeptides to be linked or conjugated to the peptide tag and/or polypeptide (peptide tag binding partner). Thus, the peptide, oligopeptide or polypeptide and the peptide tag and/or polypeptide (peptide tag binding partner) may be linked directly to each other or may be linked indirectly via one or more spacer sequences. Thus, a spacer sequence may separate or separate two or more separate portions of a recombinant or synthetic polypeptide. In some embodiments, the spacer may be at the N-terminus or C-terminus of the peptide tag and/or polypeptide (peptide tag binding partner). In some embodiments, the spacer may flank the peptide tag and/or polypeptide (peptide tag binding partner).
The precise nature of the spacer sequence is not critical and it may be of variable length and/or sequence, for example it may be of 1 to 40, more particularly 2 to 20, 1 to 15, 1 to 12, 1 to 10, 1 to 8 or 1 to 6 residues, for example 6, 7,8, 9, 10 or more residues. For example, a spacer sequence, if present, can have 1-15, 1-12, 1-10, 1-8, or 1-6 residues, and the like. The nature of the residues is not critical, they may for example be any amino acid, such as neutral or aliphatic, or they may be hydrophobic, or polar or charged or structured, such as proline. In some preferred embodiments, the linker is a serine and/or glycine rich sequence.
Thus, exemplary spacer sequences include any single amino acid residue, e.g., S, G, L, V, P, R, H, M, A or E or a di-, tri-, tetra-, penta-, or hexapeptide consisting of one or more such residues.
Thus, in some embodiments, the present invention provides a recombinant or synthetic polypeptide comprising a peptide tag and/or polypeptide (peptide tag binding partner) of the invention as defined above, i.e. a recombinant or synthetic polypeptide comprising a peptide, oligopeptide or polypeptide (e.g. a heterologous peptide, oligopeptide or polypeptide, i.e. a peptide, oligopeptide or polypeptide not normally associated with a peptide tag or polypeptide of the invention, e.g. from another biological peptide, oligopeptide or polypeptide) fused to a peptide tag and/or polypeptide (peptide tag binding partner) of the invention. The recombinant or synthetic polypeptide optionally comprises a spacer as defined above.
Recombinant or synthetic polypeptides of the invention may also comprise a purification moiety or tag to facilitate purification thereof (e.g., prior to use in the methods and uses of the invention discussed below). Any suitable purification moiety or tag may be incorporated into the polypeptide, and such moieties are well known in the art. For example, in some embodiments, a recombinant or synthetic polypeptide may comprise a peptide purification tag or portion, such as a His tag sequence. Such purification moieties or tags may be incorporated at any position within the polypeptide. In some preferred embodiments, the purification moiety is located at or near the N-or C-terminus of the polypeptide (i.e., within 5, 10, 15, 20 amino acids thereof).
As mentioned above, the advantages of the present invention result from the fact that: a peptide tag and/or polypeptide (peptide tag binding partner) incorporated into a peptide, oligopeptide or polypeptide (e.g., a recombinant or synthetic polypeptide of the invention) may be completely genetically encoded. Thus, in a further aspect, the present invention provides a nucleic acid molecule encoding a peptide tag, a polypeptide (peptide tag binding partner) or a recombinant or synthetic polypeptide as defined above.
In some embodiments, the nucleic acid molecule encoding a peptide tag as defined above comprises SEQ ID NO: 11-13 or a nucleotide sequence identical to SEQ ID NO: 11-13 has at least 80% sequence identity.
In some embodiments, the nucleic acid molecule encoding a binding partner as defined above comprises SEQ ID NO: 14 or a nucleotide sequence corresponding to SEQ ID NO: 14 has a nucleotide sequence of at least 80% sequence identity.
Preferably, the nucleic acid molecule is at least 85, 90, 95, 96, 97, 98, 99 or 100% identical to the sequence to which it is compared.
Nucleic acid sequence identity can be determined, for example, by DNA sequence determination. The FASTA search uses the GCG package with default and variable pam factors, and sets the gap creation penalty to 12.0 and the gap extension penalty to 4.0, with a window of 6 nucleotides. Preferably, the comparison is made over the entire length of the sequence, but may be made over a smaller comparison window, e.g., less than 600, 500, 400, 300, 200, 100, or 50 contiguous nucleotides.
The nucleic acid molecule of the invention may consist of: ribonucleotides and/or deoxyribonucleotides and synthetic residues such as synthetic nucleotides capable of participating in Watson-Crick type or similar base pair interactions. Preferably, the nucleic acid molecule is DNA or RNA.
The above-described nucleic acid molecules may be operably linked to expression control sequences or recombinant DNA cloning vectors or carriers comprising such recombinant DNA molecules. This allows the peptides and polypeptides of the invention to be expressed in cells as gene products, the expression of which is directed by the gene introduced into the cell of interest. Gene expression is directed by a promoter active in the target cell, and any form of linear or circular nucleic acid (e.g., DNA) vector can be inserted for integration into the genome or independent replication or transient transfection/expression. Suitable transformation or transfection techniques are well described in the literature. Alternatively, naked nucleic acid (e.g., DNA or RNA, which may include one or more synthetic residues, such as base analogs) molecules may be introduced directly into cells to produce the peptides and polypeptides of the invention. Alternatively, the nucleic acid may be converted to mRNA by in vitro transcription and the associated protein may be produced by in vitro translation.
Suitable expression vectors include appropriate control sequences, such as translation (e.g., start and stop codons, ribosome binding sites) and transcriptional control elements (e.g., promoter-operator regions, termination sequences), linked in matching reading frames to the nucleic acid molecules of the invention. Suitable vectors may include plasmids and viruses (including bacteriophages and eukaryotic viruses). Suitable viral vectors include baculovirus as well as adenovirus, adeno-associated virus, herpes and vaccinia/poxvirus. Many other viral vectors are described in the art. Examples of suitable vectors include bacterial and mammalian expression vectors pGEX-KG, pEF-neo and pEF-HA.
As mentioned above, the recombinant or synthetic polypeptides of the invention may comprise additional sequences (e.g. peptide/polypeptide tags to facilitate purification of the polypeptide) and thus the nucleic acid molecule may conveniently be fused to DNA encoding additional peptides or polypeptides, e.g. His-tags, maltose binding proteins, which upon expression may result in a fusion protein.
Thus viewed from a further aspect the invention provides a vector, preferably an expression vector, comprising a nucleic acid molecule as defined above.
Further aspects of the invention include methods of preparing a recombinant nucleic acid molecule according to the invention, comprising inserting a nucleic acid molecule of the invention encoding a peptide tag and/or a polypeptide (peptide tag binding partner) of the invention into a vector nucleic acid.
The nucleic acid molecule of the invention, preferably contained in a vector, may be introduced into a cell by any suitable means. Suitable transformation or transfection techniques are well described in the literature. A variety of techniques are known and can be used to introduce these vectors into prokaryotic or eukaryotic cells for expression. Preferred host cells for this purpose include insect cell lines, yeasts, mammalian cell lines or E.coli, for example strain BL21/
Thus, in a further aspect, there is provided a recombinant host cell comprising a nucleic acid molecule and/or a vector as described above.
"recombinant" means that the nucleic acid molecule and/or vector has been introduced into a host cell. The host cell may or may not naturally contain endogenous copies of the nucleic acid molecule, but it is recombinant in that exogenous or other endogenous copies of the nucleic acid molecule and/or vector have been introduced.
A further aspect of the invention provides a method of preparing a peptide tag and/or polypeptide (peptide tag binding partner) of the invention as defined above, the method comprising culturing a host cell containing a nucleic acid molecule as defined above under conditions comprising expression of a nucleic acid molecule encoding a peptide tag and/or polypeptide (peptide tag binding partner) as defined above, and recovering the molecule (peptide tag and/or polypeptide (peptide tag binding partner)) produced thereby. Expressed peptide tags and/or polypeptides (peptide tag binding partners) form a further aspect of the invention.
In some embodiments, the peptide tags and/or polypeptides of the invention (peptide tag binding partners), or the peptide tags and/or polypeptides used in the methods and uses of the invention, may be produced synthetically, for example by linking amino acids or smaller synthetically produced peptides, or more conveniently by recombinant expression of nucleic acid molecules encoding the above polypeptides.
The nucleic acid molecules of the invention can be produced synthetically by any suitable method known in the art.
Thus, the peptide tag and/or polypeptide (peptide tag binding partner) of the invention may be an isolated, purified, recombinant or synthetic peptide tag or polypeptide.
The term "polypeptide" is used interchangeably herein with the term "protein". As mentioned above, the term polypeptide or protein generally includes any amino acid sequence comprising at least 40 consecutive amino acid residues, such as at least 50, 60, 70, 80, 90, 100, 150 amino acids, such as 40-1000, 50-900, 60-800, 70-700, 80-600, 90-500, 100-400 amino acids. Similarly, the nucleic acid molecule of the invention may be an isolated, purified, recombinant or synthetic nucleic acid molecule.
Thus, from a further perspective, the peptide tags, polypeptides and nucleic acid molecules of the invention are preferably non-natural, i.e. non-naturally occurring molecules.
Standard amino acid nomenclature is used herein. Thus, the full name of an amino acid residue may be used interchangeably with a one-letter code or a three-letter abbreviation. For example, lysine may be substituted with K or Lys, isoleucine may be substituted with I or IIe, and the like. Furthermore, the terms aspartate and aspartate, and glutamate are used interchangeably herein and may be replaced with Asp or D or Glu or E, respectively.
Although it is envisaged that the peptide tags and polypeptides of the invention (peptide tag binding partners) and used in the invention may be produced recombinantly, which is a preferred embodiment of the invention, it will be apparent that the peptide tags and polypeptides of the invention (peptide tag binding partners) may be conjugated to proteins or other entities (e.g. molecules or components) as defined above by other means. In other words, the peptide tag or polypeptide (peptide tag binding partner) and other molecules, components or entities, such as proteins, may be combined in any suitable manner, such as by recombination, followed by conjugation (linking) to form a peptide tag-other component conjugate or polypeptide (peptide tag binding partner) -other component conjugate that may be used in the methods and uses of the invention. For example, a peptide tag and/or polypeptide (peptide tag binding partner) of the invention may be produced synthetically or recombinantly as described above and conjugated to another component (e.g., a protein) via a non-peptide linker or spacer (e.g., a chemical linker or spacer).
Thus, in some embodiments, the peptide tag and/or polypeptide (peptide tag binding partner) and other components (e.g., proteins) may be linked together directly by a bond or indirectly through a linking group. Where a linking group is used, such groups may be selected to provide covalent attachment of the peptide tag or polypeptide (peptide tag binding partner) and other entity (e.g. protein) via the linking group. The linking group of interest can vary widely depending on the nature of the other entity (e.g., a protein). When present, the linking group is biologically inert in many embodiments.
Many linking groups are known to those skilled in the art and may find use in the present invention. In representative embodiments, the linking group is typically at least about 50 daltons, typically at least about 100 daltons, and may be as large as 1000 daltons or more, for example up to 1000000 daltons if the linking group includes a spacer, but typically will not exceed about 500 daltons and typically will not exceed about 300 daltons. Typically, such linkers will comprise a spacer at either end, the spacer having a reactive functional group capable of covalently bonding to a peptide tag or binding partner and other molecules or components (e.g., proteins).
Spacers of interest may include aliphatic and unsaturated hydrocarbon chains, spacers containing heteroatoms of oxygen (ethers, e.g. polyethylene glycol) or nitrogen (polyamines), peptides, carbohydrates, cyclic or acyclic systems which may contain heteroatoms. The spacer may also be comprised of a ligand that binds to the metal such that the presence of the metal ion coordinates two or more ligands to form a complex. Specific spacers include: 1, 4-diaminohexane, xylylenediamine, terephthalic acid, 3, 6-dioxooctanedioic acid, ethylenediamine-N, N-diacetic acid, 1 '-ethylenebis (5-oxo-3-pyrrolidinecarboxylic acid), 4' -ethylenedipiperidine, oligoethylene glycol and polyethylene glycol. Potential reactive functional groups include nucleophilic functional groups (amine, alcohol, thiol, hydrazide), electrophilic functional groups (aldehyde, ester, vinyl ketone, epoxide, isocyanate, maleimide), functional groups capable of forming disulfide bonds or binding to metals by cycloaddition reactions. Specific examples include primary and secondary amines, hydroxamic acids, N-hydroxysuccinimidyl carbonates, oxycarbonylimidazoles, nitrophenyl esters, trifluoroethyl esters, glycidyl ethers, vinyl sulfones, and maleimides. Specific linker groups may find use in the capping agents of the invention, including heterofunctional compounds such as azidobenzoyl hydrazine, N- [4- (p-azidosalicylamidoamino) butyl ] -3' - [2' -pyridyldithio ] propionamide), bis-sulfosuccinimidyl suberate, dimethyl diimidate, disuccinimidyl tartrate, N-maleimidobutyryloxysuccinimidyl benzoate, N-hydroxysulfopyrimidine, N-succinimidyl [ 4-azidophenyl ] -1,3' -dithiopropionate, N-succinimidyl [ 4-iodoacetyl ] aminobenzoate, glutaraldehyde and succinimidyl-4- [ N-maleimidomethyl ] cyclohexane-1-carboxylate, N-hydroxysuccinimide ester of 3- (2-pyridyldithio) propionic acid (SPDP), N-hydroxysuccinimide ester of 4- (N-maleimidomethyl) -cyclohexane-1-carboxylic acid (SMCC), and the like. For example, the spacer may be formed from an azide reacted with an alkyne or a tetrazine reacted with a trans-cyclooctene or norbornene.
In some embodiments, it may be useful to modify one or more residues in the peptide tag and/or polypeptide (peptide tag binding partner) to facilitate conjugation of these molecules and/or to improve the stability of the peptide tag and/or polypeptide (peptide tag binding partner). Thus, in some embodiments, a peptide tag or polypeptide (peptide tag binding partner) of or for use in the present invention may comprise a non-natural or non-standard amino acid.
In some embodiments, a peptide tag or polypeptide (peptide tag binding partner) of or for use in the present invention may comprise one or more, e.g., at least 1, 2, 3, 4, 5, non-conventional amino acids, e.g., 10, 15, 20 or more, non-conventional amino acids, i.e., amino acids having a side chain not encoded by the standard genetic code, referred to herein as "non-coding amino acids" (see, e.g., table 1). These may be selected from amino acids formed by metabolic processes, such as ornithine or taurine, and/or artificially modified amino acids, such as 9H-fluoren-9-ylmethoxycarbonyl (Fmoc), (tert) -butyloxycarbonyl (Boc), 2,5,7, 8-pentamethylbenzopyran-6-sulfonyl (Pmc) -protected amino acids or amino acids with a benzyloxy-carbonyl (Z) group.
Examples of non-standard or structurally similar amino acids that may be used in the peptide tags and/or polypeptides (peptide tag binding partners) of the present invention, as well as examples of non-standard or structurally similar amino acids used in the present invention, are D amino acids, amide isosteres (e.g., N-methylamide, retro-amide, thioamide, thioester, phosphonate, ketomethylene, hydroxymethylene, fluorovinyl, (E) -vinyl, methyleneamino, methylenethio, or alkane), L-N-methylamino, D-alpha-methylamino, D-N-methylamino. Table 1 lists examples of non-conventional (i.e., non-coding) amino acids.
TABLE 1
In some embodiments, it may be useful to fuse or conjugate the peptide tags and/or polypeptides (peptide tag binding partners) of the invention to a solid phase substrate (i.e. solid phase or solid support), and it will be apparent that this may be achieved in any convenient manner. Thus, the means or means of immobilization and the solid support may be selected, depending on the choice, from any number of means of immobilization and solid supports widely known in the art and described in the literature. Thus, a peptide tag or polypeptide (peptide tag binding partner) may be directly bound to a support (e.g. chemically cross-linked), for example by a domain or portion of the peptide tag or polypeptide. In some embodiments, the peptide tag or polypeptide (peptide tag binding partner) may be indirectly bound through a linker group or through an intermediate binding group (e.g., through biotin-streptavidin interaction). Thus, the peptide tag or polypeptide (peptide tag binding partner) may be covalently or non-covalently attached to the solid support. The linkage may be a reversible (e.g., cleavable) or irreversible linkage. Thus, in some embodiments, the linkage may be cleaved, for example, enzymatically, chemically, or photo. The connection may be a light sensitive connection.
Thus, in some embodiments, a peptide tag or polypeptide (peptide tag binding partner) may provide a carrier for means of immobilization (e.g., an affinity binding partner, such as biotin or hapten, which is capable of binding to its binding partner, such as a homologous binding partner, such as streptavidin or an antibody). In some embodiments, the interaction between the peptide tag or polypeptide (peptide tag binding partner) and the solid support must be sufficiently robust to allow a washing step to be performed, i.e., the interaction between the peptide tag or polypeptide (peptide tag binding partner) and the solid support is not disrupted (significantly disrupted) by the washing step. For example, preferably less than 5%, preferably less than 4,3, 2, 1, 0.5 or 0.1% of the peptide tag or polypeptide (peptide tag binding partner) is removed or eluted from the solid phase in each washing step.
The solid support (phase or substrate) may be any known support or matrix currently widely used or proposed for immobilization, separation. These may take the form of particles (which may be, for example, magnetic, paramagnetic or non-magnetic beads), sheets, gels, filters, membranes, fibers, capillaries, slides, arrays or microtiter strips, tubes, plates or wells, and the like.
The support may be made of glass, silica, latex or polymeric material. Materials with high surface area for fusion protein binding are suitable. Such a carrier may have an irregular surface and may be, for example, porous or particulate, such as granules, fibres, a mesh, a sinter or a sieve. Particulate materials, such as beads, are useful due to their greater binding capacity, particularly polymeric beads.
Conveniently, the particulate solid support used according to the invention will comprise spherical beads. The size of the beads is not critical, but they may, for example, be of the order of at least 1 μm in diameter, preferably of the order of at least 2 μm, and their maximum diameter is preferably not more than 10, for example not more than 6 μm.
Monodisperse particles, i.e., particles of substantially uniform size (e.g., sizes with a standard deviation of less than 5% in diameter), have the advantage of providing very uniform reaction reproducibility. Representative monodisperse polymer particles may be produced by the technique described in US-A-4336173.
However, to facilitate handling and separation, magnetic beads are advantageous. As used herein, the term "magnetic" means that the carrier is capable of having an applied magnetic moment when placed in a magnetic field, and is therefore movable under the influence of that magnetic field. In other words, the carrier comprising the magnetic particles can be easily removed by magnetic aggregation, which provides a fast, simple and efficient way of separating the particles after the isopeptide bond formation step.
In some embodiments, the solid support is an amylose resin.
In another embodiment, the present invention provides a kit, in particular for use in the methods and uses of the present invention, i.e. for conjugating two molecules or components by isopeptide bond, wherein the two molecules or components in the complex are conjugated by isopeptide bond, wherein the kit comprises:
(a) a peptide (peptide tag) as defined above, optionally conjugated or fused to a molecule or component, e.g. a protein; and
(b) a polypeptide as defined above (a peptide tag binding partner) optionally conjugated or fused to a molecule or component such as a protein, e.g. a recombinant or synthetic polypeptide comprising a polypeptide as defined above (a peptide tag binding partner); and/or
(c) Nucleic acid molecules, in particular vectors, encoding the peptides (peptide tags) as defined in (a); and
(d) a nucleic acid molecule, in particular a vector, encoding a polypeptide (peptide tag binding partner) as defined in (b).
It is clear that the peptide tags and polypeptides (peptide tag binding partners) of the invention have a wide range of uses. On the other hand, the peptide tags and polypeptides (peptide tag binding partners) of the present invention can be used in a variety of industries.
For example, in some embodiments, the peptide tags and polypeptides (peptide tag binding partners) of the present invention can be used to target fluorescent or other biophysical probes or labels to specific proteins. In this regard, the protein of interest can be modified to incorporate a peptide tag of the invention (e.g., any of SEQ ID NOS: 3-5), as described above, fused or conjugated to a polypeptide (peptide tag binding partner, e.g., SEQ ID NO: 2) with a fluorescent or other biophysical probe or label. The modified protein and the probe or label may be contacted together under conditions suitable to allow spontaneous formation of isopeptide bonds between the peptide tag and the polypeptide (peptide tag binding partner), thereby labelling the protein with the label or probe via isopeptide bonds.
In some embodiments, the peptide tags and polypeptides (peptide tag binding partners) of the present invention are useful in the protein fixation of proteomics. In this regard, the protein of interest can be modified to incorporate a peptide tag of the invention (e.g., any of SEQ ID NOs: 3-5) and the solid substrate can be fused or conjugated to a polypeptide (peptide tag binding partner, e.g., SEQ ID NO: 2). The modified protein and solid substrate may be contacted together under conditions suitable to allow spontaneous formation of isopeptide bonds between the peptide tag and the polypeptide (peptide tag binding partner), thereby immobilizing the protein on the solid substrate via isopeptide bonds. It is clear that the peptide tags and polypeptides (peptide tag binding partners) of the invention can be used to simultaneously immobilize multiple proteins on a solid phase/substrate.
In further embodiments, the peptide tags and polypeptides (peptide tag binding partners) of the invention may be used to conjugate antigens to virus-like particles, viruses, bacteria or multimeric scaffolds for vaccination. For example, the production of virus-like particles, viruses or bacteria displaying a polypeptide (peptide tag binding partner) of the invention (e.g., SEQ ID NO: 2) on a surface will facilitate the conjugation of an antigen comprising a peptide tag of the invention (e.g., any of SEQ ID NO: 3-5) to its surface via a second peptide bond. In this regard, antigen multimerization elicits a strongly enhanced immune response. Thus, in some embodiments, the molecule or component fused to a polypeptide of the invention is a viral capsid protein, and/or the molecule or component fused to a peptide tag of the invention is an antigen, e.g., an antigen associated with a particular disease (e.g., infection).
In other embodiments, a peptide tag and a polypeptide (peptide tag binding partner) may be used to cyclize the enzyme, for example by fusing the peptide tag and binding partner to each end of the enzyme, and then allowing for the spontaneous formation of an isopeptide bond between the peptide tag and the polypeptide (peptide tag binding partner). In this regard, it has been demonstrated that cyclization of the enzyme increases the recovery capacity of the enzyme.
In particular, cyclization of the enzyme or enzyme polymer (fusion protein) can improve the thermostability of the protein or protein unit in the enzyme polymer. In this regard, enzymes are valuable tools in many processes, but they are unstable and difficult to recover. Enzyme polymers have greater stability to temperature, pH and organic solvents, and there is an increasing desire to use enzyme polymers in industrial processes. However, the production of enzyme polymers typically uses glutaraldehyde non-specific reactions, and this will destroy or denature (i.e., reduce its activity) many potentially useful enzymes. The use of the peptide tags and polypeptides (peptide tag binding partners) of the invention to site-specifically link proteins into chains (polymers) by isopeptide bonds is expected to enhance the resilience of enzymes, for example enzymes for use in diagnostics or for addition to animal feed. In a particularly preferred embodiment, the enzyme may be stabilized by cyclization, as described above.
The peptide tags and polypeptides (peptide tag binding partners) of the invention may also be used to link various enzymes into a pathway to promote metabolic efficiency, as described in WO 2016/193746. In this regard, enzymes often work together in pathways inside the cell, and it has traditionally been difficult to link multiple enzymes together outside the cell (in vitro). Thus, the peptide tags and polypeptides (peptide tag binding partners) of the invention can be used to couple or conjugating enzymes to produce fusion proteins and thus enhance the activity of multi-step enzymatic pathways, which can be used for a range of industrial transformations and diagnostics.
The peptide tags and polypeptides (peptide tag binding partners) of the invention may also be used in the production of antibody polymers. In this regard, antibodies are one of the most important classes of drugs, commonly used for attachment to surfaces. However, mixing of the antigens in the sample and thus capture of the antigens in the sample is ineffective near the surface. By extending the antibody chain, it is expected that capture efficiency will increase. This would be of particular value in the isolation of circulating tumor cells, which is currently one of the most promising methods for achieving early cancer diagnosis.
In another embodiment, the peptide tags and polypeptides (peptide tag binding partners) of the invention may be used in the manufacture of a medicament for activating cell signaling. In this regard, many of the most effective methods of activating cellular function are through protein ligands. In practice, however, protein ligands generally do not work alone, but rather work with specific combinations of other signal molecules. Thus, the peptide tags and polypeptides (peptide tag binding partners) of the invention allow the production of customized fusion proteins (i.e. proteomes) which can give optimal activation of cellular signals. These fusion proteins (proteomes) can be used to control cell survival, division or differentiation.
In further embodiments, the peptide tags and polypeptides (peptide tag binding partners) of the invention may be used to generate hydrogels for the culture of eukaryotic cells (e.g., neurons), stem cells, to prepare biomaterials, to antibody functionalize with dyes or enzymes, and to stabilize enzymes by cyclization.
Drawings
The invention will now be described in more detail in the following non-limiting examples with reference to the following figures:
FIG. 1 shows a schematic view of aA flow chart for panning to select spy tag variants displayed on pIII of M13 phage is shown.
FIG. 2A bar graph illustrating the quantification (mean ± 1sd, n ═ 3) of spy label phages recovered using wild-type (WT) spy capturer bait compared to the amount of spy label phages recovered after selection using non-reactive spy capturer EQ in colony forming units (cfu) is shown (a); and (B) a sequence table of spy tag variants selected from the last round of N-terminal library (NLib1-3, SEQ ID NO: 15-17) and subsequent C-terminal library (CLib1-10, SEQ ID NO: 18-27). WT refers to the sequence of the spy tag (SEQ ID NO: 6), while
FIG. 3Shows the variation of the spy capture agent with the strongest reactivity with the spy label N-terminal libraryTime course of the reaction of deletion variants of the body (NLibl-MBP). PPVPT represents SEQ ID NO: 15, PVPT represents SEQ ID NO: 30, VPT represents SEQ ID NO: 31, PT represents SEQ ID NO: 32. data are shown as mean ± 1s.d. of triplicates. Some error bars are too small to be visible.
FIG. 4A schematic diagram of (a) a phage display selection protocol for accelerated spy catcher variants is shown. Spy catcher mutants on M13 phage were panned against biotinylated Avi tag-spy tag-MBP bait before eluting TEV protease from streptavidin beads. (B) Shows that the spyware capture agent phages recovered after screening with the WT spy label-MBP or the non-reactive spy label DA-MBP control were quantified as cfu (mean ± 1s.d., n ═ 3).
FIG. 5An alignment of the amino acid sequences of the variants selected from the last round of spy catcher library selection is shown. No change: very conservative changes. Conservative changes, gaps indicate distant changes. WT represents SEQ ID NO:7, L1C1 represents SEQ ID NO: 33, L1C4 represents SEQ ID NO: 34, L1C2 represents SEQ ID NO: 35, L2C1 represents SEQ ID NO: 36, L1C3 represents SEQ ID NO: 37, L1C6 refers to SEQ ID NO: 38, L2C8 refers to SEQ ID NO: 39, SC002 refers to SEQ ID NO: 40.
FIG. 6A graph showing the reaction time course of phage selected spy catcher variants is shown. Spy tag-MBP was incubated with spy capture agent and selected variants, each protein at 1 μ M in PBS at pH7.5 at 25 ℃. After boiling, the reaction was analyzed by Coomassie stained SDS-PAGE. Data show the average of duplicate reactions. Spy capture agent refers to SEQ ID NO:7, L1C1 refers to SEQ ID NO: 33, L1C4 refers to SEQ ID NO: 34, L1C2 refers to SEQ ID NO: 35, L2C1 refers to SEQ ID NO: 36, L1C3 refers to SEQ ID NO: 37, L1C6 refers to SEQ ID NO: 38, L2C8 refers to SEQ ID NO: 39.
FIG. 7An SDS-PAGE gel is shown showing that the self-reaction of L1C6 spy catcher variants is blocked in
FIG. 8Differential scanning calorimetry is shown showing spy catcher covered with
FIG. 9An SDS-PAGE gel is shown (a) that characterizes the spontaneous isopeptide bond formation between the
FIG. 10 shows a schematic view of aA time course chart of the reaction of the spy catcher 002-sfGFP with the spy tag 002-MBP or the reaction of the spy catcher-sfGFP with the spy tag-MBP in succinate-phospho-glycine buffer at pH7.0 at (A) 1. mu.M and (B) 10. mu.M is shown. (mean ± 1s.d. of triplicates; some error bars were too small to be visible) (B).
FIG. 11A graphical representation is shown quantifying the rate constant of reaction of the
FIG. 12An SDS-PAGE gel is shown which describes the test for completion of the
FIG. 13 shows(A) A graph depicting pH dependence of a reaction of
FIG. 14A time course diagram illustrating the reaction of the MBPx-spy catcher and MBPx-
FIG. 15 shows a schematic view of aA time course graph depicting the reaction of 0.5 μ M D5T spy capturer 002(SEQ ID NO: 40) with (A)0.5 μ M spy tag 002-MBP (SEQ ID NO: 3-MBP) or spy tag 002T 3H-MBP (SEQ ID NO: 4-MBP), and (B)0.5 μ M spy tag 002-T3H-MBP (SEQ ID NO: 4-MBP) or
FIG. 16A graph depicting the rate analysis of the reaction of 0.5. mu. M
Detailed Description
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