Biomacromolecule conjugate and preparation method and application thereof
阅读说明：本技术 一种生物大分子偶联物及其制备方法和用途 (Biomacromolecule conjugate and preparation method and application thereof ) 是由 贾凌云 彭强 臧柏林 于 2021-08-17 设计创作，主要内容包括：本发明涉及生物大分子偶联物的制备方法,其包括以下步骤：(a)提供含有醛基的生物大分子,生物大分子包括蛋白质和多肽；(b)提供米氏酸的酰胺衍生物；和(c)将含有醛基的生物大分子与米氏酸的酰胺衍生物进行偶联反应,得到生物大分子偶联物。本发明还涉及具有通式I的米氏酸的酰胺衍生物和通过上述制备方法得到的生物大分子偶联物。本发明的方法反应条件温和、反应速率快、反应产物稳定,能够实现小分子化合物(药物、荧光团、聚合物)的定点偶联和蛋白质的定向固载,对蛋白质的结构和功能无不利影响。(The invention relates to a preparation method of a biomacromolecule conjugate, which comprises the following steps: (a) providing a biomacromolecule containing aldehyde groups, wherein the biomacromolecule comprises protein and polypeptide; (b) providing an amide derivative of Meldrum's acid; and (c) carrying out coupling reaction on the biomacromolecule containing the aldehyde group and the amide derivative of the Meldrum's acid to obtain the biomacromolecule conjugate. The invention also relates to amide derivatives of Meldrum's acid having the general formula I and biomacromolecule conjugates obtained by the above preparation method. The method has the advantages of mild reaction conditions, high reaction rate and stable reaction products, can realize the site-specific coupling of small molecular compounds (drugs, fluorophores and polymers) and the directional immobilization of proteins, and has no adverse effect on the structure and the function of the proteins.)
1. A method of preparing a biomacromolecule conjugate, comprising the steps of:
(a) providing a biomacromolecule containing aldehyde groups, wherein the biomacromolecule containing aldehyde groups comprises protein and/or polypeptide;
(b) providing an amide derivative of Meldrum's acid;
(c) and carrying out coupling reaction on the biomacromolecule containing the aldehyde group and the amide derivative of the Meldrum's acid to obtain the biomacromolecule conjugate.
2. The method of claim 1, wherein step (c) is performed at a pH of 2.5 to 10.5 and a temperature of-20 ℃ to 70 ℃, preferably at a pH of 3 to 10 and 4 to 9, more preferably at 4 to 7, even more preferably at 6 to 7 and most preferably at 6.5, and preferably at a temperature of-10 ℃ to 60 ℃ and at a temperature of-0 ℃ to 50 ℃, more preferably at 10 ℃ to 40 ℃ and most preferably at 25 ℃ to 37 ℃;
and the aldehyde group-containing biomacromolecule in step (a) is a protein;
the biomacromolecule conjugate package in step (c)Protein aldehyde-based conjugatesWherein the R group is a functional group coupled with protein, and comprises a fluorophore, a chemotherapeutic drug and a radiotherapeutic drug, wherein the fluorophore, the chemotherapeutic drug and the radiotherapeutic drug comprise an alkyl group with the carbon number of 1-50, an alkenyl group with the carbon number of 2-50 and/or an aryl or heteroaryl group with the carbon number of 6-50.
3. The production method according to claim 1 or 2, wherein the amide derivative of Meldrum's acid has the following general formula I:
wherein R is1Is alkyl having a carbon number of 1 to 50, cycloalkyl having a carbon number of 2 to 50, alkenyl having a carbon number of 2 to 50, alkynyl having a carbon number of 2 to 50, aryl or heteroaryl having a carbon number of 6 to 50, and the heteroatom is selected from N, O, S and P; r2Is- (CH)2)nCONHR3And n is a positive integer of 1 to 60, preferably 1 to 30, more preferably 1 to 20 and most preferably 1 to 10, R3Is hydrogen, alkyl having a carbon number of 1 to 50, cycloalkyl having a carbon number of 2 to 50, alkenyl having a carbon number of 2 to 50, alkynyl having a carbon number of 2 to 50, aryl or heteroaryl having a carbon number of 6 to 50, and the heteroatom is selected from N, O, S and P; r1、R2And R3Each may be substituted by R4Substituted, R4Is fluorine, chlorine, bromine, iodine, an alkyl group having a carbon number of 1 to 50, a cycloalkyl group having a carbon number of 2 to 50, an aryl group having a carbon number of 6 to 50 or a heteroaryl group, and the heteroatom is selected from N, O, S and P; the amide derivative of Meldrum's acid preferably comprisesWherein the R group is a functional group coupled with protein, the functional group comprises fluorophore, chemotherapeutic drug and radiotherapeutic drugThe medicine and radiotherapy medicine contain alkyl with carbon number of 1-50, alkenyl with carbon number of 2-50 and/or aryl or heteroaryl with carbon number of 6-50.
4. The production method according to any one of claims 1 to 3, wherein R is1To R3Is an alkyl group having a carbon number of 1 to 50, preferably an alkyl group having a carbon number of 1 to 30, more preferably an alkyl group having a carbon number of 1 to 15; r4Fluorine, chlorine, bromine, iodine, an alkyl group having a carbon number of 1 to 50, preferably fluorine, chlorine, bromine, iodine, an alkyl group having a carbon number of 1 to 30, more preferably an alkyl group having a carbon number of 1 to 10.
5. The production method according to any one of claims 1 to 4, wherein the amide derivative of Meldrum's acid is produced from Meldrum's acid having the following general formula II and an amide condensing agent, H2N-R3Reacting at normal temperature and normal pressure to obtain:
wherein R is5Is- (CH)2)nCOOH, n is a positive integer of 1 to 60, preferably 1 to 30, more preferably 1 to 20 and most preferably 1 to 10, and R5Can be substituted by R4Substitution; the amide condensing agent is selected from 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, O-benzotriazol-N, N, N, -tetramethylurea hexafluorophosphate, 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea hexafluorophosphate, 2-succinimidyl-1, 1,3, 3-tetramethylurea tetrafluoroborate, 2- (5-norbornene-2, 3-dicarboximidyl) -1,1,3, 3-tetramethylurea tetrafluoroborate or benzotriazol-1-yl-oxy-trispyrrolidinyl-phosphonium hexafluorophosphate, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate is preferred.
6. The preparation method according to any one of claims 1 to 5, wherein the aldehyde group-containing biological macromolecule is a protein, and preferably the protein has an amino acid sequence that is 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% and 100% identical to the amino acid sequence of SEQ ID No.1, preferably the identity is 95%, 96%, 97%, 98%, 99% and 100%, more preferably 98%, 99% and 100%, most preferably 100%; the aldehyde group of the protein comprises an aldehyde group obtained by the N-terminal oxidation reaction or amine transfer reaction of the protein, a protein aldehyde group label obtained by an enzyme method and an aldehyde group contained in an unnatural amino acid in the protein; the protein aldehyde coupling comprises coupling of a fluorophore, a chemotherapeutic drug, a radiotherapy drug, a polypeptide, a protein, a nucleic acid and an affinity tag and immobilization of the protein.
7. The preparation method according to any one of claims 1 to 6, wherein the aldehyde group-containing biomacromolecule comprises a formylglycine-containing protein obtained by interacting a cysteine-containing protein with a formylglycine-generating enzyme.
8. Use of a biomacromolecule conjugate according to any one of claims 1 to 7 in protein immobilization, wherein a biomacromolecule containing an amino group is immobilized using the Meldrum's acid of general formula II according to claim 5, the immobilization being performed by condensation of the carboxyl group of the Meldrum's acid of general formula II with the amino group of the biomacromolecule at normal temperature and pressure, the biomacromolecule being preferably a protein and a polypeptide, most preferably a protein, the immobilization support comprising agarose gel, ferroferric oxide magnetic microspheres, silica microspheres, and a metal surface.
9. An amide derivative of Meldrum's acid of the general formula I according to claim 3.
10. A biomacromolecule conjugate obtained by the production method according to any one of claims 1 to 8.
Technical Field
The invention relates to the field of biochemistry, in particular to a biomacromolecule conjugate and a preparation method and application thereof.
Background
Site-directed coupling of proteins is a key technology for preparing protein drugs, diagnostic imaging reagents and biofunctionalized materials. Due to its good bio-orthogonality, aldehyde groups are widely used as functional groups for site-directed coupling of proteins.
The existing aldehyde group modification technology mostly adopts an amino group, hydrazine group or oxyamino group-based nucleophilic reagent as an aldehyde group coupling reagent, but has the defects of violent reaction conditions, slow reaction rate, low product stability and the like. Since nucleophiles based on amino, hydrazino or oxyamino groups have a relatively small nucleophilicity (large pKa value), the reaction can only proceed relatively slowly under acidic conditions; the formed carbon-nitrogen double bond is unstable and is easily hydrolyzed under acidic conditions. In particular, patents US5633351A and EP0913691a1 disclose schiff base-based protein aldehyde-based coupling methods, but the reaction is reversible, the reaction efficiency is slow and the product is hydrolytically unstable. Other protein aldehyde-based coupling reactions generally require the presence of organic solvents and are extremely detrimental to the structure and function of proteins (see non-patent document: j.am.chem.soc.,2010,132, 9546-.
In addition, non-patent literature (org. Lett.2020April 03; 22(7): 2626-2629) reports a method for protein lysine modification based on Michael addition acceptor of Meldrum's acid derivatives. In addition, patents US5243053A and EP0206673a2 disclose the preparation of meldrum's acid derivatives and a series of derivatives. Furthermore, patent EP0578849A1 and the non-patent literature (Tetrahedron Letters,1978,19(20): 1759-.
In particular, the patents US5633351A, EP0913691a1 disclose schiff base-based protein aldehyde-based coupling methods, but the reaction is reversible, the reaction efficiency is slow and the product is hydrolytically unstable. Other protein aldehyde-based coupling reactions generally require the presence of organic solvents, which are extremely detrimental to the structure and function of the protein (see J.Am. chem.Soc.,2010,132, 9546-9548; chem. Commun.,2011,47, 9066-11068; chem. Commun.,2012,48, 11079-11081; Org.Lett.,2015,17, 1361-1364). The non-patent literature (Omar Boutureira et al, chem. Rev.2015,115,2174-2195) reviews the disadvantages of non-uniform product, large influence on protein structure and function, and poor reaction reproducibility caused by the existing non-site-specific uncontrollable modification of proteins.
Aiming at the technical problems of severe reaction conditions, low reaction efficiency, unstable products, non-uniform products, great influence on the structure and function of the protein and poor reaction reproducibility of protein aldehyde coupling strategies in the prior art, a biomacromolecule conjugate, a preparation method and application thereof are required to be developed to solve the technical problems, so that aldehyde coupling reaction conditions are mild, reaction rate is high, reaction products are stable, and controllable fixed-point modification of the protein is realized to have no adverse influence on the structure and function of the protein.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, in one aspect, the present invention provides a method for preparing a biomacromolecule conjugate, comprising the steps of:
(a) providing a biomacromolecule containing aldehyde groups, wherein the biomacromolecule containing aldehyde groups comprises protein and polypeptide;
(b) providing an amide derivative of Meldrum's acid;
(c) and (3) carrying out coupling reaction on the biomacromolecule containing the aldehyde group and the amide derivative of the Meldrum's acid to obtain the biomacromolecule conjugate.
According to a preferred embodiment of the present invention, wherein step (c) is carried out at a pH of 2.5 to 10.5 and a temperature of-20 ℃ to 70 ℃, the pH is preferably 3 to 10 and 4 to 9, more preferably 4 to 7, even more preferably 6 to 7 and most preferably 6.5, and the temperature is preferably-10 ℃ to 60 ℃ and-0 ℃ to 50 ℃, more preferably 10 ℃ to 40 ℃ and most preferably 25 ℃ to 37 ℃.
According to a preferred embodiment of the present invention, the aldehyde group-containing biopolymer in step (a) comprises a protein having aldehyde groups.
According to a preferred embodiment of the present invention, the biomacromolecule conjugate in step (c) comprises a protein aldehyde-based conjugateWherein the R group is a functional group coupled with protein, and comprises a fluorophore, a chemotherapeutic drug and a radiotherapeutic drug, wherein the fluorophore, the chemotherapeutic drug and the radiotherapeutic drug comprise an alkyl group with the carbon number of 1-50, an alkenyl group with the carbon number of 2-50 and/or an aryl or heteroaryl group with the carbon number of 6-50.
According to a preferred embodiment of the present invention, wherein the amide derivative of Meldrum's acid has the following general formula I:
wherein R is1Is alkyl having a carbon number of 1 to 50, cycloalkyl having a carbon number of 2 to 50, alkenyl having a carbon number of 2 to 50, alkynyl having a carbon number of 2 to 50, aryl or heteroaryl having a carbon number of 6 to 50, and the heteroatom is selected from N, O, S and P; r2Is- (CH)2)nCONHR3And n is a positive integer of 1 to 60, preferably 1 to 30, more preferably 1 to 20 and most preferably 1 to 10, R3Is hydrogen, alkyl having a carbon number of 1 to 50, cycloalkyl having a carbon number of 2 to 50, alkenyl having a carbon number of 2 to 50, alkynyl having a carbon number of 2 to 50, aryl or heteroaryl having a carbon number of 6 to 50, and the heteroatom is selected from N, O, S and P; r1、R2And R3Each may be substituted by R4Substituted, R4Is fluorine, chlorine, bromine, iodine, an alkyl group having a carbon number of 1 to 50, a cycloalkyl group having a carbon number of 2 to 50, an aryl group having a carbon number of 6 to 50 or a heteroaryl group, and the heteroatom is selected from N, O, S and P.
According to a preferred embodiment of the present invention, wherein the amide derivative of Meldrum's acid comprisesWherein the R group is a functional group coupled with protein, and comprises fluorophore, chemotherapeutic drug and radiotherapy drug, wherein the fluorophore, chemotherapeutic drug and radiotherapy drug comprise alkyl with carbon number of 1-50 and carbonAn alkenyl group having a number of 2 to 50 and/or an aryl or heteroaryl group having a carbon number of 6 to 50.
According to a preferred embodiment of the invention, wherein R1To R3Preferably an alkyl group having a carbon number of 1 to 50, more preferably an alkyl group having a carbon number of 1 to 30, most preferably an alkyl group having a carbon number of 1 to 15; r4Preferred are fluorine, chlorine, bromine, iodine, and an alkyl group having 1 to 50 carbon atoms, more preferred are fluorine, chlorine, bromine, iodine, and an alkyl group having 1 to 30 carbon atoms, and most preferred is an alkyl group having 1 to 10 carbon atoms.
According to a preferred embodiment of the present invention, wherein the amide derivative of Meldrum's acid is prepared by condensing Meldrum's acid having the following formula II with an amide, H2N-R3Reacting at normal temperature and normal pressure to obtain:
wherein R is5Is- (CH)2)nCOOH, n is a positive integer of 1 to 60, preferably 1 to 30, more preferably 1 to 20 and most preferably 1 to 10, and R5Can be substituted by R4Substitution; the amide condensing agent is selected from 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, O-benzotriazol-N, N, N, -tetramethylurea hexafluorophosphate, 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea hexafluorophosphate, 2-succinimidyl-1, 1,3, 3-tetramethylurea tetrafluoroborate, 2- (5-norbornene-2, 3-dicarboximidyl) -1,1,3, 3-tetramethylurea tetrafluoroborate quaternary ammonium salt or benzotriazol-1-yl-oxytripyrrolidinyl phosphorus hexafluorophosphate, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate is preferred.
According to a preferred embodiment of the invention, wherein the biological macromolecule is a protein, and preferably the protein has an amino acid sequence which is 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% and 100% identical to the amino acid sequence of SEQ ID No.1, preferably the identity is 95%, 96%, 97%, 98%, 99% and 100%, more preferably 98%, 99% and 100%, most preferably 100%.
According to a preferred embodiment of the present invention, wherein the aldehyde group of the protein includes an aldehyde group obtained by an N-terminal oxidation reaction or amine transfer reaction of the protein, an aldehyde tag of the protein obtained by an enzymatic method, and an aldehyde group contained in an unnatural amino acid in the protein.
According to a preferred embodiment of the invention, wherein the protein aldehyde-based conjugation comprises conjugation of fluorophores, chemotherapeutic drugs, radiotherapeutic drugs, polypeptides, proteins, nucleic acids, affinity tags and immobilization of proteins.
According to a preferred embodiment of the present invention, wherein the aldehyde group-containing biomacromolecule comprises a formylglycine-containing protein obtained by interacting a cysteine-containing protein with a formylglycine-generating enzyme.
In another aspect, the present invention relates to a use of the biomacromolecule conjugate in protein immobilization, wherein the amino group-containing biomacromolecule is immobilized by using the michelia acid having the general formula II, the immobilization is performed by condensing carboxyl of the michelia acid having the general formula II and amino group of the biomacromolecule at normal temperature and normal pressure, the biomacromolecule is preferably protein and polypeptide, most preferably protein, and the immobilization carrier comprises agarose gel, ferroferric oxide magnetic microspheres, silica microspheres and metal surfaces.
In addition, the present invention relates to an amide derivative of Meldrum's acid having the general formula I described above and a biomacromolecule conjugate obtained according to the above preparation method.
Effects of the invention
According to the method, the reaction conditions are mild, the reaction rate is high, the reaction product is stable, and the corresponding defects of the traditional protein aldehyde group modification method are overcome; by the method, site-specific coupling of small molecular compounds (including drugs, fluorophores and polymers) and directional immobilization of proteins can be realized, and the structure and the function of the proteins are not adversely affected.
Drawings
The invention will be further described with reference to the accompanying drawings, but the invention is not limited thereto. Wherein:
FIG. 1 shows a schematic representation of a coupling reaction of an aldehyde group-containing protein with a Meldrum's acid amide derivative according to one embodiment of the present invention.
FIG. 2A shows an electrophoresis diagram showing the expression and purification of a protein according to an embodiment of the present invention (lane M represents an electrophoretic Marker (reference standard), lane 1 is a cell body after induction of expression, lane 2 is a cell disruption supernatant, lane 3 is a cell fragment, and lane 4 is a purified protein); FIG. 2B is a graph showing the relationship between the measured molecular weight and the theoretical molecular weight of the protein of FIG. 2A prior to the introduction of aldehyde groups; FIG. 2B shows a graph of the measured molecular weight versus theoretical molecular weight for the protein of FIG. 2A after the introduction of aldehyde groups.
Fig. 3 shows a schematic representation of a process for the preparation of a meldrum's acid derivative according to one embodiment of the present invention.
FIG. 4 is a graph showing the relationship between the reaction conversion rate and pH in the reaction of a protein having an aldehyde group with a Meldrum's acid derivative under different pH conditions according to one embodiment of the present invention.
FIG. 5A is a graph showing the measured molecular weight versus theoretical molecular weight of a protein conjugate having aldehyde groups obtained at a pH of 7.0 according to one embodiment of the present invention; FIG. 2B is a graph showing the measured molecular weight of a protein conjugate having aldehyde groups obtained at a pH of 2.8 as a function of theoretical molecular weight according to another embodiment of the present invention; FIG. 2C is a graph showing the measured molecular weight of the protein conjugate having aldehyde groups obtained at pH 10.0 according to still another embodiment of the present invention as a function of the theoretical molecular weight.
FIG. 6 shows a graph of molar ellipticity versus wavelength for proteins bearing aldehyde groups before and after conjugation, according to one embodiment of the present invention.
FIG. 7 shows a schematic representation of a reaction for protein immobilization by Meldrum's acid according to one embodiment of the present invention.
Fig. 8 shows a graph of protein immobilization versus time according to an embodiment of the invention.
FIG. 9 shows a schematic representation of a coupling reaction of an aldehyde group-containing protein with a Meldrum's acid amide derivative according to another embodiment of the present invention.
FIG. 10A shows an electrophoresis diagram showing the expression and purification of a protein according to another embodiment of the present invention (lane M represents an electrophoretic Marker (reference standard), lane 1 is a cell body after induction of expression, lane 2 is a cell disruption supernatant, lane 3 is a cell fragment, and lane 4 is a purified protein); FIG. 10B is a graph showing the measured molecular weight versus theoretical molecular weight for the protein of FIG. 10A prior to the introduction of aldehyde groups; FIG. 10C shows a plot of the measured molecular weight versus theoretical molecular weight for the protein of FIG. 10A after the introduction of aldehyde groups.
Fig. 11 shows a schematic representation of efficient, highly specific recognition of tumor cells by coupling fluorophores to aldehyde group-containing proteins according to another embodiment of the present invention.
FIG. 12 shows a schematic representation of a coupling reaction of an aldehyde group-containing protein with a Meldrum's acid amide derivative according to yet another embodiment of the present invention.
FIG. 13A shows an electrophoresis chart showing the expression and purification of a protein according to still another embodiment of the present invention (lane M represents an electrophoretic Marker (reference standard), lane 1 is a cell body after induction of expression, lane 2 is a cell disruption supernatant, lane 3 is a cell fragment, and lane 4 is a purified protein); FIG. 13B is a graph showing the measured molecular weight versus theoretical molecular weight for the protein of FIG. 13A prior to the introduction of aldehyde groups; FIG. 13C shows a graph of the measured molecular weight versus theoretical molecular weight for the protein of FIG. 13A after the introduction of aldehyde groups.
FIG. 14 shows a graph of cell viability as a function of protein conjugate concentration according to one embodiment of the invention.
Fig. 15A shows a mass spectrum of a protein based on green fluorescent protein after introduction of aldehyde groups after coupling with a fluorophore according to an embodiment of the present invention; fig. 15B shows a mass spectrum of a protein based on the anti-epidermal growth factor receptor nanobody after aldehyde group introduction after fluorophore coupling according to one embodiment of the present invention; fig. 15C shows a mass spectrum of a protein after coupling of a fluorophore based on the anti- β 2 microglobulin nanobody after introduction of an aldehyde group according to one embodiment of the present invention.
Fig. 16A shows a mass spectrum of a protein based on green fluorescent protein after introduction of aldehyde groups after coupling with a chemotherapeutic drug according to an embodiment of the present invention; fig. 16B shows a mass spectrum of a protein after coupling with a chemotherapeutic drug based on the anti-egf receptor nanobody after introduction of aldehyde groups according to an embodiment of the present invention; fig. 16C shows a mass spectrum of a protein after coupling of a chemotherapeutic drug based on the anti- β 2 microglobulin nanobody after introduction of an aldehyde group according to one embodiment of the present invention.
Fig. 17A, 17B and 17C show graphs showing the changes in molecular weight of the coupling products obtained after coupling a fluorophore based on the anti-epidermal growth factor receptor nanobody after the introduction of an aldehyde group, at pH 7, pH 2.8 and pH 10.0, respectively, according to an embodiment of the present invention.
Fig. 18 is a graph showing the change of the molar ellipticity of an anti-egf receptor nanobody according to an embodiment of the present invention with respect to wavelength before and after coupling to a fluorophore.
Detailed Description
For a clearer understanding of the present invention, the concept and mechanism analysis and the like of the present invention will be described in detail below by way of specific embodiments and with reference to the accompanying drawings, but the present invention is not limited thereto.
It is to be understood that the numerical ranges herein include the endpoints and any point between the endpoints. For example, the range of values 1 to 10 includes 1, 2,3, 4, 5, 6, 7, 8, 9, 10; the numerical range of 0.1-0.9 includes 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9.
The term "biological macromolecule" as used herein refers to a macromolecule such as a protein, polypeptide, nucleic acid, polysaccharide, etc., that is present within a cell of an organism. There are thousands to hundreds of thousands of atoms in each biomacromolecule, with molecular weights ranging from tens of thousands to over millions. The biomacromolecule is mostly polymerized by simple constitutional structures, the constitutional units of protein and polypeptide are amino acids, the constitutional units of nucleic acid are nucleotides, and the constitutional units of polysaccharide are monosaccharides. From the chemical structure, the protein and polypeptide are formed by dehydration condensation of alpha-L-amino acid, the nucleic acid is formed by dehydration condensation of purine and pyrimidine bases, sugar D-ribose or 2-deoxy-D-ribose and phosphate, and the polysaccharide is formed by dehydration condensation of monosaccharide.
The term "aldehyde group-containing biopolymer" as used herein refers to the above-mentioned biopolymer containing one or more aldehyde groups.
The term "protein" as used herein is a covalent polypeptide chain formed by the condensation of amino acids end to end. Proteins include natural and synthetic proteins. Each native protein has its own unique spatial or three-dimensional structure, which is commonly referred to as the conformation of the protein, i.e., the structure of the protein. The molecular structure of proteins can be divided into four levels: (1) primary structure: linear amino acid sequences that make up a protein polypeptide chain; (2) secondary structure: stable structures, mainly alpha helices and beta sheets, formed by means of hydrogen bonds between the C ═ O and N — H groups between different amino acids; (3) tertiary structure: a three-dimensional structure of a protein molecule formed by the arrangement of a plurality of secondary structure elements in a three-dimensional space; (4) quaternary structure: are used to describe the formation of functional protein complex molecules by the interaction between different polypeptide chains (subunits). In addition to these structural levels, proteins can be switched among a number of similar structures to perform their biological functions. For functional structural changes, these tertiary or quaternary structures are usually described by chemical conformations, and the corresponding structural transformations are called conformational changes.
In the present invention, commonly used proteins include, but are not limited to: (1) green Fluorescent Protein (GFP), a β -barrel protein 1 consisting of 238 amino acids, having the amino acid sequence of SEQ ID No.1 below, with a molecular weight of about 27 kDa; GFP was isolated from the crystal jellyfish Aequorea victoria; GFP can convert the blue fluorescence emitted by aequorin through chemical action into green fluorescence 2 through energy transfer. (2) The nano anti-epidermal growth factor receptor antibody has the amino acid sequence shown in SEQ ID NO.2 and has molecular weight of about 18.00834 kDa. (3) The beta 2 microglobulin resisting nano antibody has an amino acid sequence shown in SEQ ID NO.3 and has molecular weight of 18.45833 kDa. These proteins are available from Biotechnology (Shanghai) Inc. and have the following amino acid sequences:
amino acid sequence of SEQ ID NO.1 of green fluorescent protein:
GSSHHHHHHSSGLVPRGSHMSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT GKLPVPWPTLVTTLTYGVQC FSRYPDHMKR HDFFKSAMPE GYVQERTISF KDDGTYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNFNSHNV YITADKQKNG IKANFKIRHN VEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSVLSKDPNEKRDHMVL LEFVTAAGIT HGMDELYK GGGGSLCTPSR
the amino acid sequence of the anti-epidermal growth factor receptor nano antibody SEQ ID NO. 2:
AEFQVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSEPKTPKPQPQPQPQPQPNPTTEHHHHHHGGGGSLCTPSR
the amino acid sequence of SEQ ID NO.3 of the beta 2 microglobulin resisting nano antibody is as follows:
AQVQLQESGGGSVQAGGSLRLSCAASGYTDSRYCMAWFRQAPGKEREWVARINSGRDITYYADSVKGRFTFSQDNAKNTVYLQMDSLEPEDTATYYCATDIPLRCRDIVAKGGDGFRYWGQGTQVTVSSEPKTPKPQPQPQPQPQPNPTTEHHHHHHGGGGSLCTPSR
the term "polypeptide" as used herein refers to a peptide consisting of three or more amino acid molecules, a compound formed by alpha-amino acids linked together by peptide bonds, which is an intermediate product of proteolysis. The polypeptides have a molecular weight of less than 10,000Da and are able to pass through the semi-permeable membrane without being precipitated by trichloroacetic acid and ammonium sulfate. In addition, there are documents that refer to peptides consisting of 2 to 10 amino acids as oligopeptides (also referred to as small molecule peptides); peptides consisting of 10-50 amino acids are called polypeptides; peptides consisting of more than 50 amino acids are called proteins. Any polypeptide can be used as the polypeptide to be studied in the present invention in the art, and those skilled in the art can select a polypeptide having an appropriate structure as necessary to impart a corresponding functional effect.
The term "Meldrum's acid" as used herein has the following general formula II:
wherein R is1Is alkyl having a carbon number of 1 to 50, cycloalkyl having a carbon number of 2 to 50, alkenyl having a carbon number of 2 to 50, alkynyl having a carbon number of 2 to 50, aryl or heteroaryl having a carbon number of 6 to 50, and the heteroatom is selected from N, O, S and P; r5Is- (CH)2)nCOOH, n is a positive integer of 1-60, preferably 1-30, more preferably 1-20 and most preferably 1-10; and R is1And R5Each may be substituted by R4Substituted, R4Is fluorine, chlorine, bromine, iodine, an alkyl group having a carbon number of 1 to 50, a cycloalkyl group having a carbon number of 2 to 50, an aryl group having a carbon number of 6 to 50 or a heteroaryl group, and the heteroatom is selected from N, O, S and P.
The term "amide derivative of Meldrum's acid" as used herein has the following general formula I:
wherein R is1As described above; r2Is- (CH)2)nCONHR3And n is a positive integer of 1 to 60, preferably 1 to 30, more preferably 1 to 20 and most preferably 1 to 10, R3Is hydrogen, alkyl having a carbon number of 1 to 50, cycloalkyl having a carbon number of 2 to 50, alkenyl having a carbon number of 2 to 50, alkynyl having a carbon number of 2 to 50, aryl or heteroaryl having a carbon number of 6 to 50, and the heteroatom is selected from N, O, S and P; r1、R2And R3Each may be substituted by R4And (4) substitution.
In the present invention, amide derivatives of common meldrum's acid include, but are not limited to, amide derivatives of meldrum's acid having, for example, the following structure:
(wherein, the R group may be of any functionMolecules, such as fluorophores, drugs, etc.);
the term "coupling reaction" as used herein is the process of obtaining an organic (bio) molecule by performing a chemical reaction on two organic (bio) chemical units. The chemical reaction herein includes a reaction of a grignard reagent with an electrophile, a reaction of a lithium reagent with an electrophile, electrophilic and nucleophilic reactions on an aromatic ring, and the like. The coupling reaction herein generally refers to an aldehyde-based coupling reaction of a protein or polypeptide.
According to the present invention, the aldehyde group of the protein or polypeptide includes an aldehyde group obtained by an N-terminal oxidation reaction or amine transfer reaction of the protein or polypeptide, an aldehyde group tag of the protein or polypeptide obtained by an enzymatic method, and an unnatural amino acid containing an aldehyde group in the protein or polypeptide. In addition, aldehyde-based conjugation of proteins or polypeptides according to the present invention includes conjugation of fluorophores, chemotherapeutic drugs, radiotherapeutic drugs, polypeptides, proteins, nucleic acids, affinity tags, and immobilization of proteins. The solid carrier comprises agarose gel, ferroferric oxide magnetic microspheres, silicon dioxide microspheres and a metal surface.
For other terms of the present invention, those skilled in the art will generally understand their ordinary meaning in the art, unless otherwise indicated.
The Michelson acid and aldehyde group reaction belongs to common organic synthesis reactions in the field, but does not give corresponding suggestion to the invention, which is the first unexpected discovery of the inventor. The reason is as follows:
first, the type of Meldrum's acid derivatives mentioned in the present invention has not been reported in the literature, but only one 1, 3-dioxane-4, 6-dione derivative in which two groups attached to the carbon atom of the ring 2 are C, respectively, is disclosed in patent EP0578849A11-5Alkyl or phenyl and hydrogen or C1-4An alkyl group.
Secondly, although the michelia acid and its derivatives and aldehyde group reaction have a great deal of application in the field of organic synthesis, the reaction with protein aldehyde group has not been reported. The structure of the Meldrum's acid is used as a synthetic raw material in organic synthesis, and the Meldrum's acid structure in the invention provides a functional group which reacts with aldehyde group of protein, thereby realizing functional derivation of the protein, therefore, the function of the Meldrum's acid structure in the organic synthesis is obviously different from that in the invention.
Moreover, due to the special microenvironment and steric hindrance of the protein, the aldehyde group properties of the protein are greatly different from those of small molecules, so that the aldehyde group reaction in organic synthesis cannot give suggestion to the aldehyde group reaction of the protein. For example, aldehyde and amino groups in organic synthesis react very rapidly and a catalyst can be used to increase the reaction rate, whereas aldehyde and amino groups in proteins react very slowly and a catalyst has no effect (see non-patent document: J.Am.chem.Soc.2011, 133, 16127-16135). Therefore, since the aldehyde-based coupling reaction of the biomacromolecule (including protein and polypeptide) of the present invention has insurmountable technical obstacles according to the conventional organic synthesis reaction, the advantageous effects achieved by the present invention are hardly expected from the prior art.
In particular, protein conjugates play a key role in many areas. For example, protein-coupled imaging molecules are used for targeted imaging and specific recognition in the field of biological imaging; in the field of tumor treatment, antibody molecule coupled chemotherapeutic drugs are used for targeted killing of tumors; in the field of biological detection, protein is immobilized on a carrier to prepare a protein chip, so that high-flux and high-specificity target object detection is realized. The main challenge of protein conjugation at present is mainly how to ensure that the structure and function of the protein are not damaged while protein conjugation is achieved, and to ensure the uniformity and reproducibility of the conjugate. The traditional protein coupling usually adopts side chain groups of amino acids (such as lysine and cysteine) endogenous to the protein as coupling groups, but because the amino acids are widely existed in the protein, the random coupling of the protein can cause the problems that the structure and the function of the protein are easily affected, the uniformity of the conjugate is poor, the reproducibility is poor, and the like. While aldehyde groups are not common in proteins, site-directed coupling of proteins can be achieved by introducing aldehyde groups in specific positions of the protein and using them as coupling groups. The site-specific coupling of the protein can overcome the defects of the traditional protein coupling method, thereby affecting the structure and the function of the protein to the minimum extent and ensuring the uniformity and the reproducibility of the conjugate. In contrast to the present invention, most of the existing aldehyde group modification techniques employ a nucleophilic reagent based on an amino group, a hydrazine group or an oxyamino group as a coupling reagent for aldehyde group, but they have the disadvantages of severe reaction conditions, slow reaction rate, low product stability, etc. In this regard, the present invention develops a novel protein aldehyde-based coupling strategy as described herein.
Through the above unexpected discovery of the present inventors, the disadvantages of the prior art, such as the technical problems of the prior protein aldehyde coupling strategy, such as severe reaction conditions, low reaction efficiency, unstable product, etc., as described above, are solved; realizes the controllable fixed-point modification of the protein, and overcomes the technical problems of non-uniform product, large influence on the structure and the function of the protein, poor reaction reproducibility and the like in the prior art.
For the same reason as described above, the present inventors have also unexpectedly found for the first time a novel use for immobilizing a protein using a carrier having a Meldrum's acid structure. The advantages of protein immobilization are: protein immobilization can improve the stability of protein, is beneficial to the separation and recovery and reutilization of the protein, or endows a certain biological function to the material. Common examples are: immobilized enzymes, antibody chips, and the like.
In addition, for the same reasons as above, the present inventors have also unexpectedly found for the first time that the protein aldehyde-based coupling method can be applied to protein-modifying compounds (e.g., fluorophores, chemotherapeutic drugs, radiotherapeutic drugs, polypeptides, proteins, nucleic acids, and affinity tags).
Preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings, but the scope of the present invention is not limited thereto.
Aldehyde group coupling reaction
As shown in FIG. 1, according to a preferred embodiment of the present invention, a protein having an aldehyde group is subjected to a coupling reaction with a Meldrum's acid amide derivative. In particularProteins with aldehyde groupsWith Meldrum's acid amide derivatives(wherein, R group can be any functional molecule, such as fluorophore, drug, etc.) aldehyde group coupling reaction is carried out at the pH of 2.5-10.5 and the temperature of-20 ℃ -70 ℃. In the reaction process, the peripheral electrons of the carbon atom of the No.2 ring of the 1, 3-dioxane-4, 6-diketone of the Meldrum's acid amide derivative are attracted by the carbonyl oxygen and ether bonding oxygen on the ring and the amide group on the side chain of the ring to carry positive charges, then the carbon atom of the No.2 ring with the positive charges attacks the carbonyl on the aldehyde group on the protein, and finally the protein aldehyde group conjugate is obtained
In particular, for the michelia acid amide derivatives, R is a common functional group coupled to proteins, including fluorophores, chemotherapeutic drugs, radiotherapeutic drugs, and the like. For example, these structures of fluorophores and chemotherapeutic agents typically contain alkyl groups, benzene rings, double bonds, and the like. The number of alkyl groups directly bonded to the amide bond is not less than 1, and is preferably a positive integer of 1 to 60 and 1 to 30, more preferably 1 to 20 and most preferably 1 to 10.
In fact, as long as it has the parent nucleus of the Meldrum's acid structureNamely, the aldehyde coupling reaction can be carried out with the biomacromolecule with aldehyde group, such as protein or polypeptide.
Moreover, the reaction conditions of the aldehyde coupling reaction are mild, and the reaction temperature is in the range of-20 ℃ to 70 ℃, preferably-10 ℃ to 60 ℃ and-0 ℃ to 50 ℃, more preferably 10 ℃ to 40 ℃ and most preferably 25 ℃ to 37 ℃ in view of the aldehyde coupling conversion rate of the protein; the pH is in the range of 2.5 to 10.5, preferably 3 to 10 and 4 to 9, more preferably 4 to 7, further preferably 6 to 7 and most preferably 6.5. Compared with other aldehyde coupling methods, the reaction has the advantages of mild reaction conditions, high reaction rate and stable coupling product. In addition, the time is 2 to 24 hours, preferably 4 to 24 hours, more preferably 6 to 24 hours, and most preferably 12 to 24 hours from the viewpoint of the aldehyde coupling conversion rate of the protein.
Relationship between aldehyde coupling conversion and pH
The aldehyde coupling conversion of the proteins at different pH is shown in FIG. 4. As can be seen from FIG. 4, the pH ranges corresponding to the aldehyde coupling conversion rates of the proteins from high to low are as follows: 6 to 7, 5 to 6, 4 to 5, 7 to 8 and 8 to 9.
Effect of aldehyde coupling reaction on Structure and function of protein
In addition, as can be seen from fig. 5, no significant hydrolysis of the resulting protein aldehyde conjugate occurred, whether under acidic, neutral or basic pH conditions, as can be confirmed from the measured molecular weights of the resulting conjugates at different pH and the relationship between the measured molecular weights and the theoretical molecular weights at the same pH (see fig. 5A, 5B and 5C and fig. 17A, 17B and 17C).
In addition, the effect of the coupling of proteins with aldehyde groups to the Meldrum's acid derivative on protein structure and function can be determined by circular dichroism spectroscopy. Specifically, the protein conjugate product was diluted into 10mM phosphate buffer at a final concentration of 0.03mg/ml and added to a 1mM cuvette for circular dichroism spectrum scanning. Circular dichroism determination method the Instrument used is a MOS-500 circular dichroism spectrometer (Bio-Logic Science Instrument); the operation parameters are that the spectrum scanning wavelength range is 190nm-250nm, and the scanning step length is 1 nm. As shown in fig. 6 and 18, the obtained circular dichroism spectra did not change significantly before and after the aldehyde group coupling, indicating that the coupling reaction of the protein having an aldehyde group with the michelia acid derivative did not have a significant effect on the protein structure. Therefore, the coupling reaction of the protein having aldehyde groups with the Meldrum's acid derivative does not adversely affect the structure and function of the protein.
Immobilization of proteins
In the invention, common immobilized carriers comprise agarose gel, ferroferric oxide magnetic microspheres, silicon dioxide microspheres and metal surfaces. According to a preferred embodiment of the present invention, the present invention synthesizes the immobilized agarose gel carrier containing the michelia acid structure by using a solid phase synthesis method (see fig. 7, the reaction conditions are typically normal temperature and pressure, and the pH is 6.5).
Specifically, the synthesis precursor, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide were dissolved in a dimethyl sulfoxide/phosphate buffer (pH 7.4)1:1, and (2) dissolving the mixture in the solution. The solution was added to the amino activated gel and mixed well by tumbling at room temperature. After the reaction is completed, the gel is washed with water, 1M sodium chloride solution and water in sequence, and the gel is stored in 20% ethanol solution and left to stand for later use.
The immobilization carrier was washed with an immobilization buffer. The aldehyde group-bearing protein was dissolved in MES buffer (pH 6.5) and added to the immobilization carrier. The mixture was shaken up at room temperature under reverse rotation and sampled periodically. The amount of immobilized protein was determined by measuring the difference in protein concentration in the supernatant before and after immobilization by the BCA method. The protein immobilization was measured at different time points (see FIG. 8).
The protein is immobilized, so that the stability of the protein can be improved, the separation, the recovery and the reutilization of the protein are facilitated, or a certain biological function is endowed to the material.
Introduction of aldehyde groups into proteins
As for the protein having aldehyde groups, it is formed by introducing aldehyde groups into the protein. There are many ways to introduce aldehyde groups into proteins, including but not limited to aldehyde groups obtained by N-terminal oxidation or transamination of proteins, aldehyde tags of proteins obtained by enzymatic methods, and aldehyde groups contained in unnatural amino acids in proteins.
For example, aldehyde groups are produced by using a formylglycine generating enzyme. Specifically, the formylglycine generating enzyme converts cysteine in the protein to formylglycine, thereby generating aldehyde groups, as shown in the following figure:
it can be seen that the molecular weight was reduced by about 18 when aldehyde groups were successfully introduced into the protein, i.e., after the protein without aldehyde groups was converted into the protein with aldehyde groups. Therefore, by determining whether the amount of decrease in molecular weight is about 18 (e.g., 17 to 19), it can be confirmed whether or not aldehyde groups are successfully introduced into the protein.
As shown in fig. 2A to 2C, fig. 2A is an expression purification electrophoretogram of green fluorescent protein (described in examples below) for confirming successful production of green fluorescent protein. FIG. 2B is a further confirmation that the green fluorescent protein produced is correct from a molecular weight perspective, where the observed molecular weight (29765.79) matches the theoretical molecular weight (29766.32). Furthermore, it was confirmed that aldehyde groups have been successfully introduced into the protein by the amount of change obtained by comparing the molecular weights of fig. 2B with those of fig. 2C (29765.79-29747.61 being 18.18 for the amount of change in measured molecular weight and 29766.32-29748.22 being 18.1 for the amount of change in theoretical molecular weight).
Preparation of Meldrum's acid derivatives
As shown in FIG. 3, according to a preferred embodiment of the present invention, Meldrum's acid is added in the presence of an amide condensing agent (e.g., 2- (7-azabenzotriazole) -N, N, N ', N ' -tetramethyluronium hexafluorophosphate) at normal temperature and pressureAnd H2N-R is subjected to condensation reaction to obtain the Meldrum's acid amide derivative
Amide condensing agent
In general, amidation reaction is difficult to occur, and a condensing agent is added to accelerate the reaction. The reaction principle is that carboxyl is activated first, and then the carboxyl reacts with amine to obtain amide.
As the amide condensing agent, the following four kinds of amide condensing agents are commonly used: (1) active esters, such as Carbonyldiimidazole (CDI). (2) Carbodiimide-based condensing agents such as Dicyclohexylcarbodiimide (DCC), Diisopropylcarbodiimide (DIC) and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDCI). (3) Onium salt condensing agents, which are classified into two types, mainly, a carbonium salt and a phosphonium salt; for example, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU), 6-chlorobenzotriazole-1, 1,3, 3-tetramethyluronium Hexafluorophosphate (HCTU), 2-succinimidyl-1, 1,3, 3-tetramethyluronium tetrafluoroborate (TSTU), 2- (5-norbornene-2, 3-dicarboximidyl) -1,1,3, 3-tetramethyluronium tetrafluoroborate (TNTU), O- (7-azabenzotriazole-1-yl) -bis (tetrahydropyrrolyl) carbenium hexafluorophosphate (HAPyU), O- (benzotriazol-1-yl) -N, N, N ', N' -dipyrrolyl urea Hexafluorophosphate (HBPYU), benzotriazol-1-oxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), Hexamethylphosphoramide (HMPA), benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), etc. (4) Examples of the organophosphorus condensing agents include diethyl cyanophosphate (DECP), bis (2-oxo-3-oxazolidinyl) phosphorylidene chloride (BOP-Cl), and the like. (5) Other condensing agents, for example, triphenylphosphorus-polyhalomethane, triphenylphosphorus-hexachloroacetone, triphenylphosphorus-NBS, 3-acyl-2-thiothiazoline, tris (2, 6-dimethoxyphenyl) bismuth, and the like.
In the present invention, the amide condensing agent usually used may be selected from 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, O-benzotriazol-N, N, N, -tetramethyluronium hexafluorophosphate, 6-chlorobenzotriazole-1, 1,3, 3-tetramethyluronium hexafluorophosphate, 2-succinimidyl-1, 1,3, 3-tetramethyluronium tetrafluoroborate, 2- (5-norbornene-2, 3-dicarboximidyl) -1,1,3, 3-tetramethyluronium tetrafluoroborate or benzotriazol-1-yl-oxytripyrrolidinyl phosphonium hexafluorophosphate, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate is preferred.
Measurement method
(I) electrophoresis measurement:
1. a15% separation gel was prepared according to Table 1 below. After the gel was completely separated, the water was poured off and the residual water was blotted with clean filter paper. 4 percent of concentrated glue is prepared and is immediately inserted into a clean comb to avoid generating bubbles. After the condensed gel is condensed, the comb is pulled out. And (3) installing an electrophoresis tank, and adding a proper amount of Tris-glycine electrophoresis buffer solution into the upper tank and the lower tank respectively. Avoiding the generation of bubbles at the bottom of the rubber.
TABLE 1 separation gel and concentrated gel formulations
Reagent
Separating glue (15%)
Concentrated gum (4%)
Solution A
7.5mL
1.34mL
Liquid B
3.75mL
0mL
C liquid
0ml
2ml
Ultrapure water
3.75mL
4.6mL
10% ammonium persulfate
75μL
60μL
TEMED
5μL
10μL
Total volume
15mL
8mL
2. To 100. mu.l of the electrophoresis sample, 25. mu.l of 5 Xloading buffer was added, and the mixture was subjected to boiling water bath for 10 min. Add 5. mu.l of electrophoresis sample to each well of the gel.
3. And (3) opening the electrophoresis apparatus, using the voltage of the concentrated gel section to be 92V, increasing the voltage to 120V after the bromophenol blue indicator enters the separation gel, ending electrophoresis when the bromophenol blue indicator reaches the bottom of the separation gel, closing the power supply, and taking out the gel.
4. Dyeing and decoloring: washing the gel surface after electrophoresis with double distilled water, pouring a dyeing solution (the formulas of the dyeing solution and the decoloring solution are shown in the following table 2), and placing in a water bath kettle at 90 ℃ for 20-30 min; then, the mixture was rinsed with double distilled water and poured into a decolorization solution, and decolorized on a balanced decolorization shaker, and then subjected to photographic analysis using a gel imaging system ChemiDoc XRS + (BIORAD, USA).
TABLE 2 dyeing liquor and destaining liquor formulas
Reagent
Dyeing liquid
Decolorizing liquid
Coomassie brilliant blue R-250
1g
0g
Industrial ethanol
0ml
300ml
Isopropanol (I-propanol)
250mL
0mL
Industrial acetic acid
100ml
100ml
Single distilled water
650mL
600mL
(II) circular dichroism spectrum measurement:
the conjugate was centrifuged through an Amicon 15 ml ultrafiltration centrifuge tube, 4500g, to replace the background solution with 10mM phosphate buffer (pH 7.4) at a protein concentration of 0.3 mg/ml. Mu.l of the protein solution was added to a 1mm optical path cuvette and placed in a multifunctional circular dichroism MOS-500 (BioLogic Science Instruments, France) to scan a circular dichroism spectrum between 190 and 250nm with a scanning step of 1nm and a slit width of 5 nm. Molar ellipticity was calculated using the circular dichroism spectral ratios measured by analysis using Biokine in combination with Dicroprot software, molar ellipticity at different wavelengths was calculated and plotted (e.g. figure 6). Comparison of the changes in the curves before and after coupling can lead to the fact that the coupling has no effect on the conformation of the protein.
(iii) definition of molar ellipticity and correlation between molar ellipticity and circular dichroism determination:
the raw CD spectral data obtained when testing a circular dichroism spectrum in an experiment is generally expressed in milli-degrees mdeg (Y-axis). For proteins, the molar ellipticity is generally used in the literature as the ordinate and is given in units of deg.cm2Dmol-1. Molar ellipticity relative to originalThe mdeg unit of (a) takes into account the optical path length of the cuvette, the concentration of the protein, and the number of amino acid residues of the protein, and is calculated as follows:
where X is the raw data measured by circular dichroism spectroscopy in medg, l is the optical path length, c is the concentration of the protein, and m is the total number of amino acid residues of the protein. The molar ellipticity is taken into account for all experimental parameters, so that the data obtained under different experimental conditions are comparable. The conversion in the experiment was achieved by Biokine in combination with Dicroprot software.
(tetra) relationship of circular dichroism spectra and protein structure:
the peptide bond of the protein has circular dichroism in the detection wavelength range of 190-250nm, and the circular dichroism spectrogram of the protein can reflect the change of the secondary structure of the protein.
Examples
The present invention is described in detail below by way of examples. However, the present invention is not limited to these embodiments, and those skilled in the art can make various modifications, changes, and variations within the scope of the present invention.
In the following examples, unless otherwise indicated, the methods are conventional; unless otherwise specified, the materials and reagents are commercially available.
Example 1: introduction of aldehyde group into green fluorescent protein
The aldehyde-labeled green fluorescent protein (protein sequence shown in SEQ ID NO.1, synthesized by Biotechnology (Shanghai) Co., Ltd.) was synthesized and cloned into pET28a (+) expression vector, then transformed into E.coli T7Shuffle (DE3), and cultured and induced to express in Terrific Broth medium. The cells were collected by centrifugation at 8000 Xg for 5 minutes and resuspended in disruption buffer (20mM phosphate, pH 7.4, 500mM sodium chloride, 20mM imidazole). Using a high pressure homogenization disruptor (AH-NANO, ATS Engineering limited)Cell disruption was performed, and cell disruption supernatant and cell debris were collected by centrifugation at 8000 Xg for 30 minutes. Purifying cell disruption supernatant protein with metal ion affinity chromatography column HisTrap HP 5ml (GE healthcare), and using purified FGEUltra-1510K ultrafiltration tubes (Millipore) were concentrated to 10mg/ml by ultrafiltration and pipetted into phosphate buffered saline (pH 7.4) and frozen at-80 ℃ until use. As shown in FIG. 2A, the successful expression of the target protein was confirmed by SDS-PAGE electrophoretic analysis of the cells after induction expression, cell disruption supernatant, cell debris and purified protein. The correct expression of the target protein was confirmed by high-resolution LC-MS analysis (measured molecular weight: 29765.79, theoretical molecular weight: 29766.32, shown in Table 7 below).
0.1mM aldehyde-labeled green fluorescent protein was added to triethanolamine buffer (50mM triethanolamine, 50mM sodium chloride, 2mM dithiothreitol, 0.01mM copper sulfate, pH 9.0), and 0.01mM formylglycine generating enzyme was added and incubated at room temperature for 24 h. After the reaction is completed, useThe green fluorescent protein exchange solution was concentrated to phosphate buffered saline (pH 7.4) in an Ultra-1510K ultrafiltration tube (Millipore) and frozen at-80 ℃ until use. As shown in FIGS. 2B and 2C, Table 7, the successful introduction of aldehyde groups was determined by high resolution LC-MS (measured molecular weight: 29747.61, theoretical molecular weight: 29748.22).
Amino acid sequence of SEQ ID No. 1:
GSSHHHHHHSSGLVPRGSHMSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT GKLPVPWPTLVTTLTYGVQC FSRYPDHMKR HDFFKSAMPE GYVQERTISF KDDGTYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNFNSHNV YITADKQKNG IKANFKIRHN VEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSVLSKDPNEKRDHMVL LEFVTAAGIT HGMDELYK GGGGSLCTPSR
example 2: coupling reaction of Meldrum's acid derivative and aldehyde group-containing green fluorescent protein
(1) Preparation of Meldrum's acid derivatives
The synthesis route of the Meldrum's acid derivative is shown in FIG. 3. The synthesis precursor (99 mg) and 2- (7-azabenzotriazole) -N, N' -tetramethyluronium hexafluorophosphate (190.12 mg) were dissolved in 2ml dimethylformamide, 66.07 μ l 2,4, 6-trimethylpyridine was added and stirred at room temperature for 15 minutes. In addition, the compound H with amino group2N-R (wherein R is a fluorophore) was dissolved in 1 ml of dimethylformamide, 132.15. mu.l of 2,4, 6-collidine was added, and this solution was slowly added to the reaction solution and stirred for reaction for 2 hours. Purifying by silica gel column chromatography and preparative liquid chromatography to obtain the product.
(2) Coupling reaction of Meldrum's acid derivative and protein with aldehyde group
0.1mM of green fluorescent protein having aldehyde group was dissolved in 50mM MES buffer at room temperature, 1mM of Meldrum's acid derivative was added and the reaction was carried out at various pH conditions for 3 hours. The product was analyzed by high resolution liquid chromatography-mass spectrometry. As shown in fig. 4, the michelia acid has the highest coupling efficiency (as expressed by coupling conversion) under near neutral conditions (pH 6.5). The aldehyde coupling conversion rate of green fluorescent protein as a function of pH is shown in Table 3.
TABLE 3 summary of experimental data on aldehyde coupling conversion and pH for green fluorescent protein
pH range
Aldehyde coupling conversion of proteins
pH 4-5
68-72％
pH 5-6
75-79％
pH 6-7
76-83％
pH 7-8
28-34％
pH 8-9
3-10％
As can be seen from Table 3 above, the pH is preferably from 4 to 9, more preferably from 4 to 7 and most preferably from 6 to 7, particularly 6.5, from the viewpoint of the aldehyde coupling conversion rate of the protein.
The obtained coupling product of the Meldrum's acid derivative and the aldehyde group of the protein was subjected to ultrafiltration to acidic (pH 2.8), neutral (pH 7.0) and basic (pH 10.0) solutions, incubated at room temperature for 48 hours, and it was determined whether hydrolysis occurred in the coupling product by high-resolution liquid chromatography-mass spectrometry. As shown in fig. 5A, 5B and 5C, the aldehyde-based coupling product did not undergo significant hydrolysis under acidic, neutral and basic conditions.
In addition, the effect of the coupling of aldehyde groups to the Meldrum's acid derivative on the protein structure was determined by circular dichroism spectroscopy. The conjugated protein product was diluted into 10mM phosphate buffer at a final concentration of 0.03mg/ml and added to a 1mM cuvette for circular dichroism spectroscopy. As shown in fig. 6, the obtained circular dichroism spectrum did not change significantly before and after the reaction, indicating that the coupling of aldehyde groups and the michelia acid derivative did not have a significant effect on the protein structure.
In addition, 0.1mM of aldehyde group-carrying green fluorescent protein was dissolved in 50mM MES (pH 6.5) buffer, 1mM of Meldrum's acid derivative was added and the reaction was carried out under different temperature conditions for 3 hours. The product was analyzed using a linear ion trap-high resolution liquid chromatography/mass spectrometer LTQ Orbitrap XL (available from Thermo Scientific technologies, ltd.). The aldehyde coupling conversion rate of green fluorescent protein with temperature change is shown in Table 4.
TABLE 4 summary of experimental data on aldehyde coupling conversion of green fluorescent protein versus temperature
As can be seen from Table 4 above, the temperature is preferably from-10 ℃ to 60 ℃ and from-0 ℃ to 50 ℃, more preferably from 10 ℃ to 40 ℃ and most preferably from 25 ℃ to 37 ℃ from the viewpoint of the aldehyde coupling conversion rate of the protein.
In addition, 0.1mM of green fluorescent protein having aldehyde group was dissolved in 50mM MES buffer (pH 6.5), 1mM of Meldrum's acid derivative was added and the reaction was carried out at room temperature for various times. The product was analyzed using a linear ion trap-high resolution liquid chromatography/mass spectrometer LTQ Orbitrap XL (available from Thermo Scientific technologies, ltd.). The aldehyde coupling conversion of green fluorescent protein with time is shown in Table 5.
TABLE 5 summary of experimental data on aldehyde coupling conversion of green fluorescent protein versus time
Time (hours)
Yield (%)
2
55
4
76
6
89
12
100
24
100
As can be seen from Table 5 above, the time is preferably 2 to 24 hours, more preferably 4 to 24 hours and 6 to 24 hours, and most preferably 12 to 24 hours, from the viewpoint of the aldehyde coupling conversion rate of the protein.
Example 3: immobilization of proteins using a carrier with a Meldrum's acid structure
As shown in FIG. 7, immobilized agarose gel containing a Meldrum's acid structure was synthesized by a solid phase synthesis method. 25 mg of the synthesis precursor, 0.77 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1.15 mg of N-hydroxysuccinimide were dissolved in 2ml of a 1:1 solution of dimethyl sulfoxide/phosphate buffer (pH 7.4). This solution was added to 1g of amino-activated gel, and stirred and mixed well at room temperature for 2.5 hours. After the reaction was completed, the gel was washed with 10-fold volume of water, 1M sodium chloride solution, and water in this order, and the gel was stored in 20% ethanol solution and stored at 4 ℃ for later use.
0.1g of the immobilization carrier was washed with 10 volumes of immobilization buffer (50mM MES, pH 6.5). Green fluorescent protein (0.2ml, 7mg/ml) with aldehyde groups was dissolved in MES buffer (pH 6.5) and added to the immobilization carrier. The mixture was shaken up at room temperature under reverse rotation and sampled periodically. The amount of immobilized protein was determined by measuring the difference in protein concentration between the supernatants before and after immobilization by the BCA method. The protein immobilization measured at different time points is shown in figure 8 and table 6 below.
TABLE 6 summary of experimental data for protein immobilization at different time points
Time (hours)
Protein immobilization amount (mg/g carrier)
Immobilization efficiency (%)
1
1.7
13
3
3.1
24
6
3.6
29
12
4.4
33
24
5.4
40
36
6.3
50
As is clear from table 6 and fig. 8 above, protein immobilization can improve the stability of proteins, facilitate separation and recovery of proteins, or impart a certain biological function to materials. Examples of the conventional protein immobilization include an immobilized enzyme and an antibody chip.
Example 4: aldehyde group is introduced into nano antibody of anti-epidermal growth factor receptor
The procedure of the corresponding method was the same as in example 1 except that the green fluorescent protein in example 1 was replaced with an anti-epidermal growth factor receptor nanobody (protein sequence shown in SEQ ID NO.2, synthesized by Biotechnology, Shanghai, Inc.) and a Meldrum's acid derivative was prepared as shown in FIG. 9. And (3) carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis on the thalli, cell disruption supernatant and cell fragments after induction expression and the purified protein to determine the successful expression of the target protein. The correct expression of the target protein was confirmed by high-resolution LC-MS analysis (measured molecular weight: 18008.34, theoretical molecular weight: 18008.58, as shown in Table 7 and FIG. 10 below). In addition, successful introduction of aldehyde groups was determined by high resolution LC-MS (measured molecular weight: 17990.99, theoretical molecular weight: 17990.58, as shown in Table 7 below and FIG. 10).
The amino acid sequence of SEQ ID NO. 2:
AEFQVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSEPKTPKPQPQPQPQPQPNPTTEHHHHHHGGGGSLCTPSR
example 5: coupling of Meldrum's acid derivative and anti-epidermal growth factor receptor nano antibody with aldehyde group and use of antibody conjugate in cell imaging
1. Coupling:
the anti-epidermal growth factor receptor nano antibody can target tumor cells with high expression of epidermal growth factor receptors, and efficient and high-specificity recognition of the tumor cells can be realized by coupling a fluorophore to the protein. In this experiment, a michelia acid derivative with a fluorescent group was added to an anti-epidermal growth factor receptor nanobody solution with an aldehyde group to react, as shown in fig. 9. The reaction conditions are as follows: pH 6.5, room temperature, reaction time 12 hours. After the reaction was completed, the reaction solution was added to Amicon 15 ml ultrafiltration centrifuge tubes, and the solution was changed by centrifugation at 4500g to replace the conjugate product in phosphate buffer at pH 7.4. The protein molecular weights before and after coupling were determined by a linear ion trap-high resolution liquid chromatograph-mass spectrometer LTQ Orbitrap XL (available from Thermo Scientific technologies Co., Ltd.), and the results coincided with the theoretical molecular weights (theoretical molecular weight before coupling: 18538.22, measured molecular weight: 18537.74; theoretical molecular weight after coupling: 18458.33, measured molecular weight after coupling: 18458.99).
The stability of the above-described anti-epidermal growth factor receptor nanobody-coupled product at various pHs was verified in the same manner as in example 2 (pH of FIG. 17A was 7, pH of FIG. 17B was 2.8, pH of FIG. 17C was 10.0; also shown in Table 10 below). From the above, at pH 7.4, the molecular weight of the anti-epidermal growth factor receptor nanobody coupling product was found to be 18458.99, while at pH 7, 2.8 and pH 10.0, the molecular weight of the coupling product was found to be 18537.68, 18537.49 and 18537.99, respectively. Therefore, the actually measured molecular weight change of the anti-epidermal growth factor receptor nano antibody coupling product under different pH values has no obvious change; likewise, the theoretical molecular weight of the coupled product does not change significantly at different pH's. This indicates that the above-mentioned coupled product of the anti-epidermal growth factor receptor nanobody is stable in structure at different pH values, and its function is not adversely affected.
In the same manner as in example 2, it was verified by circular dichroism that the anti-epidermal growth factor receptor nanobody had no effect on the antibody structure before and after coupling (as shown in fig. 18).
2. Cell imaging:
the amount is about 5 multiplied by 104The epidermal growth factor receptor-highly expressed tumor cell a431 cells were inoculated in DMEM medium (supplemented with 1% penicillin and streptomycin, and 10% fetal bovine serum) and cultured overnight in a cell culture incubator. The conjugate product (concentration 1. mu.M) was added to the medium and incubated at 37 ℃ for 1 hour. The cells were then washed twice with phosphate buffer and imaged using a laser scanning confocal microscope (Olympus FV1000) at an excitation wavelength of 405 nm. As shown in fig. 11, the coupling product enables targeted imaging of tumor cells with high expression of epidermal growth factor receptor.
Example 6: aldehyde group is introduced into beta 2 microglobulin resisting nano antibody
The procedure was the same as in example 1, except that the green fluorescent protein in example 1 was replaced with a nanobody against β 2 microglobulin (protein sequence shown in SEQ ID No.3, synthesized by bio-engineering (shanghai) corporation). And (3) carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis on the thalli, cell disruption supernatant and cell fragments after induction expression and the purified protein to determine the successful expression of the target protein. The correct expression of the target protein was confirmed by high-resolution LC-MS analysis (measured molecular weight: 18458.99, theoretical molecular weight: 18458.33, shown in Table 7 and FIG. 13 below). In addition, successful introduction of aldehyde groups was confirmed by high resolution LC-MS (measured molecular weight: 18439.73, theoretical molecular weight: 18440.33, as shown in Table 7 and FIG. 13 below).
Amino acid sequence of SEQ ID No. 3:
AQVQLQESGGGSVQAGGSLRLSCAASGYTDSRYCMAWFRQAPGKEREWVARINSGRDITYYADSVKGRFTFSQDNAKNTVYLQMDSLEPEDTATYYCATDIPLRCRDIVAKGGDGFRYWGQGTQVTVSSEPKTPKPQPQPQPQPQPNPTTEHHHHHHGGGGSLCTPSR
example 7: coupling of Meldrum's acid derivative and aldehyde group-containing anti-beta 2 microglobulin nano antibody and use of antibody conjugate in aspect of chemotherapeutic drugs
1. Coupling:
the anti-epidermal growth factor receptor nano antibody can target tumor cells with high expression of epidermal growth factor receptors, and can realize the target killing of the tumor cells by coupling chemotherapeutic drugs. The coupling reaction is shown in FIG. 12, and the reaction process is the same as in example 5. The protein molecular weights before and after coupling were determined by a linear ion trap-high resolution liquid chromatograph-mass spectrometer LTQ Orbitrap XL (available from Thermo Scientific technologies Co., Ltd.) and the results were matched with the theoretical molecular weights (theoretical molecular weight before coupling: 18538.22, measured molecular weight: 18537.74; theoretical molecular weight after coupling: 18700.25, measured molecular weight after coupling: 18700.52; as shown in FIG. 13).
2. Tumor cell killing:
the amount is about 5 multiplied by 104The epidermal growth factor receptor-highly expressed tumor cell A431 cells of (1% penicillin and streptomycin were added to the culture medium) were inoculated in DMEM mediumAnd 10% fetal bovine serum) and added to a 96-well plate. After overnight incubation in the incubator, different concentrations (0.02-2.5. mu.M) of conjugate were added to each well and incubation in the incubator was continued overnight. MTT (Thiazoline) was dissolved in phosphate buffer at a concentration of 5mg/ml and added in 200. mu.l per well in 96-well plates. The cells were further placed in the incubator for 4 hours, after which the medium was decanted and 200. mu.l dimethyl sulfoxide (DMSO) was added and the absorbance at 570nm was measured for each well using a microplate reader (Bio-Rad microplate reader). Cell viability was calculated from the following equation:
the cell survival curve is drawn as shown in figure 14, and the conjugate can effectively realize the target killing of the tumor cells with high expression of epidermal growth factor receptors.
The detection principle of the MTT method for determining the cell survival rate is summarized as follows: succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-violet crystalline Formazan (Formazan) and deposit in the cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and an enzyme linked immunosorbent assay detector is used for measuring the light absorption value of the formazan at the wavelength of 570nm, so that the quantity of living cells can be indirectly reflected. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number.
In addition, fig. 15 shows mass spectra of proteins based on green fluorescent protein after introduction of aldehyde groups, an anti-epidermal growth factor receptor nanobody, and an anti- β 2 microglobulin nanobody, respectively, after coupling with a fluorophore; table 8 below shows a summary of the molecular weight data of proteins before and after coupling with fluorophores, respectively, based on the aldehyde group-introduced green fluorescent protein, the anti-epidermal growth factor receptor nanobody, and the anti- β 2 microglobulin nanobody; table 9 below shows a summary of the molecular weight data of proteins based on the aldehyde group-introduced green fluorescent protein, the anti-epidermal growth factor receptor nanobody, and the anti- β 2 microglobulin nanobody before and after coupling with a chemotherapeutic drug, respectively.
TABLE 7 summary of molecular weight data for proteins before and after aldehyde group introduction for Green fluorescent protein, anti-epidermal growth factor receptor Nanobody, and anti-beta 2 microglobulin Nanobody, respectively
TABLE 8 summary of molecular weight data for proteins based on aldehyde group-introduced Green fluorescent protein, anti-epidermal growth factor receptor Nanobody, and anti- β 2 microglobulin Nanobody before and after coupling with fluorophore
TABLE 9 summary of molecular weight data of proteins before and after coupling with chemotherapeutic drugs based on aldehyde group-introduced green fluorescent protein, anti-epidermal growth factor receptor nanobody, and anti-beta 2 microglobulin nanobody
TABLE 10 stability data for anti-EGF receptor nanobody-coupled fluorophore products at different pH
pH
Theoretical molecular weight
Measured molecular weight
7.0
18538.22
18537.68
2.8
18538.22
18537.49
10.0
18538.22
18537.99
Preferred embodiments of the present invention have been described and illustrated in detail above with reference to specific examples. It should be understood by those skilled in the art that the present invention is not limited to the preferred embodiments and specific examples described above. Those skilled in the art can modify, substitute, or change the embodiments of the present invention without departing from the spirit and scope of the present invention, which also overcomes the technical problems that the present invention is directed, and achieves the objects and advantageous effects of the present invention.
Sequence listing
<110> university of Large Community
<120> biomacromolecule conjugate, preparation method and application thereof
<130> 2021-08-16
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 268
<212> PRT
<213> Artificial Sequence
<400> 1
Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro Arg
1 5 10 15
Gly Ser His Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro
20 25 30
Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val
35 40 45
Arg Gly Glu Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys
50 55 60
Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val
65 70 75 80
Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His
85 90 95
Met Lys Arg His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val
100 105 110
Gln Glu Arg Thr Ile Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg
115 120 125
Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu
130 135 140
Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu
145 150 155 160
Glu Tyr Asn Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln
165 170 175
Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp
180 185 190
Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly
195 200 205
Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser
210 215 220
Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu
225 230 235 240
Glu Phe Val Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr
245 250 255
Lys Gly Gly Gly Gly Ser Leu Cys Thr Pro Ser Arg
260 265
<210> 2
<211> 166
<212> PRT
<213> Artificial Sequence
<400> 2
Ala Glu Phe Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln
1 5 10 15
Thr Gly Gly Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser
20 25 30
Arg Ser Tyr Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
35 40 45
Glu Phe Val Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala
50 55 60
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
65 70 75 80
Thr Val Asp Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile
85 90 95
Tyr Tyr Cys Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr
100 105 110
Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu
115 120 125
Pro Lys Thr Pro Lys Pro Gln Pro Gln Pro Gln Pro Gln Pro Gln Pro
130 135 140
Asn Pro Thr Thr Glu His His His His His His Gly Gly Gly Gly Ser
145 150 155 160
Leu Cys Thr Pro Ser Arg
165
<210> 3
<211> 168
<212> PRT
<213> Artificial Sequence
<400> 3
Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Asp Ser Arg
20 25 30
Tyr Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp
35 40 45
Val Ala Arg Ile Asn Ser Gly Arg Asp Ile Thr Tyr Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Phe Ser Gln Asp Asn Ala Lys Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asp Ser Leu Glu Pro Glu Asp Thr Ala Thr Tyr Tyr
85 90 95
Cys Ala Thr Asp Ile Pro Leu Arg Cys Arg Asp Ile Val Ala Lys Gly
100 105 110
Gly Asp Gly Phe Arg Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Gln Pro Gln Pro Gln Pro
130 135 140
Gln Pro Asn Pro Thr Thr Glu His His His His His His Gly Gly Gly
145 150 155 160
Gly Ser Leu Cys Thr Pro Ser Arg
165
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