阅读说明：本技术 Crm197蛋白质表达 (CRM197 protein expression ) 是由 刘理成 卓逸明 邱家鸿 于 2019-01-18 设计创作，主要内容包括：本发明关于在细胞中生产CRM197重组蛋白质的方法。该方法包括培养包含具有多核苷酸的表达质粒的细胞,并诱导该CRM197蛋白质的表达。(The present invention relates to a method for producing a CRM197 recombinant protein in a cell. The method comprises culturing a cell comprising an expression plasmid having a polynucleotide and inducing expression of the CRM197 protein.)

1. A method of producing a CRM197 protein, comprising:

culturing a cell comprising an expression plasmid having a polynucleotide, wherein the polynucleotide comprises:

(a) a CRM197 nucleotide sequence at least 90% identical to SEQ ID NO 1, and

(b) a secretion signal sequence encoding the amino acid of SEQ ID NO 2 at the 5' end of the CRM197 nucleotide sequence; and

expression of the CRM197 protein was induced.

2. The method of claim 1, wherein the secretion signal sequence is a pectate lyase b (pelb) sequence.

3. The method of claim 2, wherein the PelB sequence is from an Erwinia species.

4. The method of claim 1, wherein the expression plasmid comprises a phosphate regulated promoter.

5. The method of claim 4, wherein the phosphate regulated promoter is a bacterial alkaline phosphatase A promoter.

6. The method of claim 1, wherein the cell comprises E.

7. The method of claim 1, wherein the CRM197 protein is soluble.

8. The method of claim 1, wherein the CRM197 protein is periplasmic.

9. The method of claim 1, wherein the CRM197 protein is folded and comprises an internal disulfide bond.

10. The method of claim 1, wherein the expression temperature of CRM197 protein is first incubated at 37 ℃ and then reduced to about 20-25 ℃.

11. The method of claim 1, wherein the inducing is performed in storage buffer at pH 7.0-8.0.

12. The method of claim 11, wherein the storage buffer is selected from the group consisting of MOPS buffer, Tris buffer, and ammonium buffer.

13. A polynucleotide comprising:

(a) a CRM197 nucleotide sequence at least 90% identical to SEQ ID NO 1, and

(b) a secretory signal sequence encoding the amino acid of SEQ ID NO 2 at the 5' end of the CRM197 nucleotide sequence.

14. An expression plasmid comprising:

the polynucleotide of claim 13; and

an inducible promoter to initiate expression of the polynucleotide.

15. The expression plasmid of claim 14, wherein the expression plasmid is deposited at 2017 at 11/28 th under ATCC accession No. PTA-124609.

16. A host cell comprising the polynucleotide of claim 13 or the expression plasmid of claim 14.

17. A CRM197 recombinant protein comprising:

(a) a polypeptide that is at least 90% identical to the amino acid sequence transcribed from the nucleotide of SEQ ID NO. 1, and

(b) 2 at the N-terminus of the polypeptide.

Technical Field

The present disclosure relates to the field of recombinant protein production in bacterial hosts. In particular, the invention relates to a method for obtaining a well-folded soluble recombinant CRM197 protein from a bacterial cell. The disclosure also relates to an expression plasmid and a host cell of the recombinant CRM197 protein.

Background

Diphtheria Toxin (DT) is a proteinaceous exotoxin synthesized by pathogens, such as Corynebacterium diphtheriae (Corynebacterium Diphtheria). The toxigenic strain contains a phage lysogen carrying the virulence gene. Diphtheria Toxin (DT) is synthesized as a 535 amino acid polypeptide containing fragment a (the catalytic domain) and fragment B (the receptor binding and transmembrane domain) linked together by disulfide bonds. The toxin binds to a cellular receptor (HB-EGF-receptor) and enters the cell via endocytosis, wherein the fragment A is released from the fragment B via proteolytic cleavage. Fragment A inhibits protein translation by catalyzing the transfer of ADP-ribosyl to N-1 of diptheria amide (diphTHamide) at histidine 715 in mammalian elongation factor 2 (EF-2). Thus, Diphtheria Toxin (DT) can kill eukaryotic cells by binding to cell surface receptors, entering the cytosol, and inactivating ribosomal elongation factor 2 (EF-2).

Cross-reactive 197 (CRM 197) gene from mutant phage beta 197^tox-Is carried. CRM197 is a non-toxic form of diphtheria toxin that contains a single amino acid substitution of glutamic acid to glycine (G52E) and loses its ADP-ribosyltransferase activity but retains binding activity. CRM197 is commonly used as a carrier in polysaccharide or oligosaccharide conjugated vaccines. It is an ideal carrier for conjugate vaccines against capsular bacteria. Conjugate vaccines comprising CRM197 covalently conjugated to a poorly immunogenic and T cell independent capsular polysaccharide produce conjugated antigens that are highly immunogenic and result in long term immunity to the antigen. It has been widely used as a carrier protein in several Vaccines, for example, Hib vaccine (Novartis Vaccines), hepta-and 13-valent pneumococcal vaccine (Wyeth) and meningococcal serogroup C-conjugated vaccine (Novartis Vaccines and Wyeth). Recent studies have shown thatCRM197 inhibits heparin-binding epidermal growth factor (HB-EGF), an Epidermal Growth Factor Receptor (EGFR) ligand. EGFR and its ligands are involved in cell development, proliferation and differentiation and can induce tumor formation.

In summary, CRM197 is a commonly used carrier protein in a combination vaccine. Its high purity, homogeneous structure and availability of lysine residues, enables the production of well-defined and characterized glycoconjugate vaccines. These attractive features of CRM197 have been used to develop a variety of CRM 197-based conjugated vaccines with proven immunogenicity.

Disclosure of Invention

In the present application, a novel approach is provided to generate a properly folded soluble CRM197 protein that forms with the correct disulfide bonds, e.g., 2 internal disulfide bonds.

In one aspect, provided herein is a method of producing a CRM197 protein. The method comprises the steps of culturing a bacterial cell comprising an expression plasmid having a polynucleotide; and inducing expression of the CRM197 protein. The polynucleotide comprises: (a) a CRM197 nucleotide sequence that is at least 90% identical to SEQ ID No. 1, and (b) a secretion signal sequence encoding the amino acid of SEQ ID No. 2 at the 5' end of the CRM197 nucleotide sequence.

Preferably, the secretion signal sequence may be a pectate lyase B (PelB) sequence. More preferably, the PelB sequence is from an Erwinia species (Erwinia spp.).

Preferably, the expression plasmid may comprise a phosphate regulated promoter. More preferably, the phosphate regulated promoter may be a bacterial alkaline phosphatase a promoter.

Preferably, the bacterial cell may comprise Escherichia coli. More preferably, the E.coli may be BL21 competent cells.

Preferably, the CRM197 protein may be soluble, periplasmic and/or properly folded. More preferably, the CRM197 protein may comprise internal disulfide bonds.

Preferably, the expression temperature of the CRM197 protein is first incubated at 37 ℃ and then reduced to about 20-25 ℃.

Preferably, the induction is carried out in storage buffer at pH 7.0-8.0.

Preferably, the storage buffer is selected from the group consisting of MOPS buffer, Tris buffer and ammonium buffer.

In another aspect, provided herein is a polynucleotide. The polynucleotide comprises: (a) a CRM197 nucleotide sequence that is at least 90% identical to SEQ ID No. 1, and (b) a secretion signal sequence encoding the amino acid of SEQ ID No. 2 at the 5' end of the CRM197 nucleotide sequence.

In another aspect, provided herein is an expression plasmid. The expression plasmid contains the polynucleotide and an inducible promoter for promoting the expression of the polynucleotide.

In another aspect, provided herein is a host cell. The host cell comprises the polynucleotide as described above or the expression plasmid as described above.

In another aspect, provided herein is a CRM197 recombinant protein. The protein was produced from the expression plasmid deposited at 28.11.2017 under ATCC accession No. PTA-124609.

In another aspect, provided herein is a CRM197 recombinant protein. The protein comprises (a) a polypeptide that is at least 90% identical to the amino acid sequence transcribed from the nucleotide sequence of SEQ ID NO:1, and (b) a secretion signal sequence of SEQ ID NO:2, located at the N-terminus of the polypeptide.

Other aspects of the invention will be apparent in view of the attached drawings and the following description.

Drawings

FIG. 1 shows the gene and amino acid sequence encoding CRM 197. The underlined sequences are 5 '-HindIII and 3' -EcoRI restriction sites.

FIG. 2 shows a plasmid map of pelBs-CRM 197_ pEGm-phoA 1.0.

Figure 3 shows an agarose gel image of the CRM197 construct. Five constructs were confirmed by restriction digestion with HindIII and EcoRI and analyzed on a 1% agarose gel. The correct construct should release about 1.67kb of the CRM197 gene fragment. Constructs No. 3-14 were further submitted for DNA sequencing and confirmed with the correct CRM197 coding sequence.

FIG. 4 shows an SDS-PAGE analysis of CRM197 expression. (A) CRM197 constructs No. 3-14 from figure 3 were transformed into competent cells BL21 and screened for CRM197 expression. Six transformants were examined under the same expression conditions. The same amount of total soluble form protein (6. mu.g) was analyzed by 10% SDS-PAGE. A strongly induced protein band was observed at 58 kDa. All these transformants were shown to have comparable CRM197 expression. Strains No. 3-14-8 were selected for further protein quality analysis. (B) Periplasmic expression of PelB-CRM197 after 28 and 45 hours of incubation. Arrows indicate CRM197 fusion proteins.

FIG. 5 shows the expression of CRM197 fused to a PelB signal sequence in E.coli BL21 strain in various media.

FIG. 6 shows the expression of CRM197 fused to a PelB signal sequence in E.coli BL21 strain at various pH values.

FIG. 7 shows the results of the cleavage site analysis of recombinant pelBs-CRM 197. (A) RP-HPLC profiling of CRM 197. One-step DEAE-purified CRM197 from the expression strains No. 3-14-8 was subjected to LC-MS analysis to confirm the PelB signal sequence and the cleavage site between CRM 197. The above results show that only the first 10 amino acids of CRM197 (1-GADDVVDSSK-10) were detected from both samples (phoA and MTB as basal media), indicating that the PelB signal sequence was accurately and completely removed by cellular protease. (B) CID spectrum of peptide at RT 25.7. The CID spectrum of the peptide was used to confirm that the peaks in 7A (1-GADDVVDSSK-10) were indeed the first 10 amino acids of CRM 197.

Figure 8 shows the results of CRM197 disulfide bond linkage analysis. (A) phoA-based medium; (B) based on MTB medium. One-step DEAE purified CRM197 from expression strains No. 3-14-8 was subjected to LC-MS analysis to check if CRM197 formed the correct disulfide bond between Cys186-Cys201 and Cys461-Cys 471. The results showed that 2 correct disulfide bonds were detected (T44-S-S-T45 and T18-T19-S-S-T22-T23), and no free thiol and disturbed disulfide bonds were identified from this 2 samples.

Figure 9 shows the results of CRM197 bacterial strain stability testing. (A) Results of protein expression stability analysis. Generations 6, 66 and 96 of CRM197 expression strains No. 3-14-8 were collected and these were subjected to CRM197 expression under the same conditions. The same amount of total soluble form protein (6. mu.g) was analyzed by 10% SDS-PAGE. Strong and comparable levels of CRM197 expression were observed in the 6 th, 66 th and 96 th generation cells after 27.5 hours, but not at 4 hours of inoculation. (B, C) results of plasmid restriction enzyme mapping analysis. Restriction enzyme digestion of pelBs-CRM 197_ pEGm phoA 1.0 plasmid extracted from strain Nos. 3-14-8 after passage 6, 66 and 96. 0.5. mu.g of DNA was digested with HindIII and EcoRI (B) or EcoRV (C) at 37 ℃ for 1 hour and further analyzed with 1% agarose gel.

Detailed Description

The foregoing and other aspects of the present disclosure will now be described in more detail with respect to other embodiments described herein. It is to be understood that the present invention may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

As used herein, the terms "comprising," "comprises," "including," "includes," "having," "has," "having," "contains," "containing," "characterized by" or any other variation thereof, are intended to cover a non-exclusive inclusion, subject to any limitation explicitly indicated. For example, a composition, mixture, process, or method that comprises a list of features is not necessarily limited to only those features but may include other features not expressly listed or inherent to such composition, mixture, process, or method.

The conjunction "consisting of" excludes any feature, step, or ingredient not specified. Except for impurities normally associated therewith, which, if in the claims, exclude materials other than the recited materials. The word "consisting of" when appearing in a clause of the text of a claim, rather than immediately after the preamble, limits only the features specified in the clause; other features are not excluded from the overall claims.

The conjunction "consisting essentially of" is used to define a composition, method that includes materials, steps, features, components, or elements in addition to those literally disclosed, provided that such additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristics of the claimed invention. The term "consisting essentially of" occupies an intermediate zone between "including" and "consisting of.

If applicants have defined an invention, or a portion thereof, as having open-ended terms, such as "comprising," it should be readily understood (unless otherwise specified) that this description should be interpreted to also use the term "consisting essentially of, or" consisting of.

As used herein, the term "about" is used to indicate a numerical value including, for example, the inherent variation of the error of a measuring device or method used to determine the value, or the variation that exists between study subjects. Generally, the term is meant to include variations of about or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, depending on the particular situation.

The use of the term "or" in the claims is intended to mean "and/or" unless explicitly indicated to refer to alternatives only or to alternatives being mutually exclusive, although the disclosure supports the definition of alternatives only and "and/or".

As used herein, the terms "transformation", "transformed" or "introducing a nucleic acid into a host cell" refer to the use of any method of introducing an exogenous nucleic acid (e.g., a vector or plasmid) into a host cell, with or without accompanying substances. The term "transformed cell" or "transformed cell" means that a foreign nucleic acid is introduced into the cell or a daughter cell thereof such that the host cell contains the foreign nucleic acid. Once introduced into the host cell, the nucleic acid is integrated into the chromosome and becomes a fragment thereof, or is retained as an extrachromosomal element, for replication purposes. Transformation of a suitable host cell with, for example, an expression vector can be accomplished using methods known in the art, such as electroporation and particle bombardment, or using chemical methods, such as catalysis of the transformation process with calcium phosphate. These methods are described, for example, in Maniatis et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, 1982), or Ausubel et al, Current Protocols in Molecular Biology (John Wiley and Sons, 1994).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for all teachings relating to the sentences and/or paragraphs that present the reference.

Embodiments of the invention according to the present application relate to methods for producing recombinant CRM197 proteins.

Methods according to embodiments of the invention involve culturing bacterial cells comprising an expression plasmid having a polynucleotide. After culture, the cells, e.g., E.coli, were induced to express the CRM197 protein under optimal conditions, thereby successfully achieving overexpression of the recombinant CRM197 protein in soluble form. Preferably, the cells are propagated or induced at a temperature of about 15 ℃ to about 37 ℃, more preferably 20-35 ℃, 20-30 ℃, or 20-25 ℃. This method will facilitate the production and purification of recombinant CRM197 in its native conformation and disulfide bonds.

The method may further comprise the step of cloning the codon optimised polynucleotide into an expression vector to transform a bacterial cell, such as an e. The polynucleotide comprises a CRM197 nucleotide sequence that is at least 90% identical to SEQ ID NO. 1, and a secretion signal sequence encoding the amino acid of SEQ ID NO. 2 at the 5' end of the CRM197 nucleotide sequence. The pectate lyase b (pelb) sequence may be used as a leader sequence for the CRM197 protein, making the CRM197 protein expressed by the cell soluble and intracellular, periplasmic or secreted. In one embodiment, the PelB sequence may be from an erwinia species. The secretory signal sequence may be directly linked to the CRM197 nucleotide sequence. Alternatively, a spacer may be present between the secretion signal sequence and the CRM197 nucleotide sequence. The spacer may comprise more or less than 9 nucleotides, for example 5 to 20 nucleotides. In a preferred embodiment, the expression enhancer comprises a ribosome binding site upstream of the CRM197 nucleotide sequence and an ATG codon.

Embodiments of the present invention will be further illustrated by the specific examples set forth below. It will be understood by those skilled in the art that these examples are for illustration only and not for the purpose of limitation, as variations and modifications may be made without departing from the scope of the invention.

Without further elaboration, it is believed that one skilled in the art can, based on the description above, utilize the present invention to its fullest extent. The following specific examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference.

Examples

Example 1: construction of pEGm-pho A1.0 vector, an expression vector derived from the alkaline phosphatase A promoter

Briefly, the codon optimized sequence of the target gene CRM197 is fused to various 5' signal sequences that can direct the expression of CRM197 to the periplasm for proper disulfide bond formation and proper protein folding. The recombinant CRM197 is operably linked to an expression control promoter under conditions suitable for expression. In related aspects, the 5' signal sequence encodes a Sec-dependent signal sequence, e.g., PelB fused to CRM197 for periplasmic expression. The optimized sequence (CRM197_ pMK-RQ) of the CRM197 gene (SEQ ID NO:1) was synthesized in the pMK-RQ vector and the signal sequence was inserted into the 5' end of CRM197 in the pMK-RQ vector using several pairs of primers. This original CRM197 sequence was optimized and fused to various signal sequences and then subcloned into an expression vector, such as pEGm-phoA 1.0, which contains a selectable marker gene, such as kanamycin, and a phosphate regulated promoter to control protein expression.

In particular, from commercially available carriersThe modification pEGm-phoA 1.0. In particular, theThe selection marker Amp of (1)^RQuilt Kan^RAnd (4) substitution. In addition, the original pTAC promoter was also replaced by the E.coli alkaline phosphatase A (phoA) promoter (Craig et al, 1991). The phoA promoter in the pEGm-phoA 1.0 expression vector was used to regulate the expression of CRM197 in media with fine control of phosphate concentration.

Example 1-1: construct kanamycin resistance gene as selectable marker

Using pET27b (+) plasmid as a template, a DNA fragment of kanamycin resistance gene was amplified and obtained with a pair of specific primers (Table 1, SEQ ID NO:3 and SEQ ID NO: 4). Also useAs template, the vector backbone of pEGm-phoA 1.0, but not Amp, was amplified with 2 specific primers (Table 1, SEQ ID NO:5 and SEQ ID NO:6)^RA resistance gene. The 2 DNA fragments were amplified using Phusion high fidelity PCR kit and further usedThe 2 DNA fragments were cloned using the seamless cloning and assembly kit (SEAMLESS cloning and Assembly) to generate a DNA fragment carrying Kan^RModification of genesVector, namely

TABLE 1

^#The sequence in the grey region is the corresponding nucleotide sequence of the PelB sequence.

Examples 1 to 2: the pTAC promoter was replaced by the phoA promoter.

Will be provided withFurther used as template to amplify the entire vector with the phoA promoter sequence insert via 2 primers (Table 1, SEQ ID NO:7 and SEQ ID NO: 8). The phoA promoter contains the Pho-box sequence in the-35 (CTGTCATAAAGTTGTCAC) region (Craig et al, 1991). Finally, after DNA sequencing, we obtained 2 new constructs with the correct phoa promoter sequence. These new expression vectors were named pEGm-pho A1.0 #2 and pEGm-pho A1.0 # 3.

Example 2: construct of CRM197 expression plasmid

Example 2-1: CRM197 gene synthesis

To be provided with(Life technologies^TM) The coding sequence of the CRM197 gene was synthesized and codon usage was optimized for e.coli expression (fig. 1). The synthetic CRM197 gene was inserted into the pMK-RQ vector, designated CRM197_ pMK-RQ, which had 2 restriction enzyme sites for further cloning, 5 '-HindIII and 3' -EcoRI.

Example 2-2: insertion of PelB Signal sequence at the 5' end of CRM197

To achieve correct folding and disulfide bond formation of CRM197 during protein synthesis, a leader nucleotide sequence of PelB (pelBs) was inserted 5' to the CRM197 coding sequence by amplifying the CRM197_ pMK-RQ with 2 designed primers (Table 1, SEQ ID NO:9 and SEQ ID NO: 10). PelB is a 22 amino acid peptide (MKYLLPTAAAGLLLLAAQPAMA; SEQ ID NO:2) that can direct the fusion protein to the periplasmic space and is removed by endogenous signal peptidases. The new CRM197 construct was named pelBs-CRM 197_ pMK-RQ.

Examples 2 to 3: construction of pelBs-CRM 197 to pEGm-phoA 1.0

The final step in the construction of the CRM197 expression plasmid was the subcloning of the pelBss-CRM197 gene into the pEGm-phoA 1.0 vector through HindIII and EcoRI restriction enzyme sites to generate the expression plasmid pelBss-CRM197_ pEGm-phoA 1.0 (fig. 2). We obtained several clones with the appropriate size of CRM197 insert, approximately 1.67 kb. Constructs 3-14 were confirmed by sequencing to have the correct pelBs-CRM 197 coding sequence and subsequently used for protein expression (FIG. 3).

Example 3: expression bacterial strain screening

The expression vector pelBs-CRM 197_ pEGm-phoA 1.0#3-14 was transformed into E.coli BL21 strain and incubated overnight at 37 ℃ on LB agar plates (containing 30. mu.g/mL kanamycin) to obtain several single colony transformants. 6 transformants were screened to select the best CRM197 expressing strain. The transformant was first inoculated into 5mL of LB medium (containing 30. mu.g/mL kanamycin) in a 14mL polypropylene tube, and cultured at 37 ℃ and 225rpm for 5 hours until OD₆₀₀About 1.0. Next, 0.5mL of each strain of the culture was transferred to 50mL of expression medium in a 250mL flask and cultured at 37 ℃ at 225rpm for another 3 hours. The expression medium contained 30mM ammonium sulfate; 2.4mM trisodium citrate dehydrate; 14.35mM potassium chloride; 1.07g/L yeast extract; 3.22g/L tryptone, 0.11M MOPS pH 8; 0.55% glucose and 0.007mM magnesium sulfate. When OD is reached₆₀₀To about 0.5-1, preferably 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0, the temperature becomes about 16 ℃ to 25 ℃, preferably about 20 ℃ to 25 ℃, more preferably 20, 21, 22, 23, 24 or 25 ℃, for 29 hours for expression of CRM 197. Tong (Chinese character of 'tong')The extent of expression of soluble CRM197 was further analyzed by SDS-PAGE to determine candidate bacterial strains. A total of 6. mu.g of each sample from the culture supernatant fraction was loaded on 10% SDS-PAGE for analysis (FIG. 4 (A)). The CRM197 expression levels among these six strains appeared to be comparable, and strains No. 3-14-8 were selected for further study.

In addition, FIG. 4(B) shows the protein induced 28 and 45 hours PelB-CRM197 expression. The expressing bacterial strains were cultured under the above conditions, but the induction times were 28 and 45 hours, respectively. From the results, protein expression was similar at 28 and 45 hours of protein induction, indicating that PelB-CRM197 expression could peak at or before 28 hours of induction.

In addition, strains Nos. 3-14-8 were deposited at The American Type Culture Collection (ATCC) at 28.11.2017, at Deliver No. 20110, USA, Va, Marina, University Daodle No. 10801 (10801University Boulevard, Manassas, VA 20110, USA), ATCC accession No. PTA-124609, and at 7.12.2017, at Food Industry Development Institute (Food Industry Research and Development Institute, FIRDI), Deliver No. 300, Taiwan, New bamboo Food route No. 331, BCRC accession No. BCRC 940662.

Example 3-A: optimization of Induction conditions

Example 3-A-1: expression medium

The periplasmic expression of PelB-CRM197 was tested in media with various buffer compositions, such as 1M MOPS, Tris or phosphate buffer. Strain Nos. 3-14-8 was inoculated and cultured in a medium, and 1M MOPS, Tris and phosphate buffer, pH8, were added. The cultured bacteria were harvested at different time points, i.e. 3 and 24 hours, to examine the effect of the buffer composition on the degree of CRM197 expression. Harvested cells were disrupted with lysis buffer. Mu.g of total soluble protein was fractionated by 10% SDS-PAGE.

The results in figure 5 show that MOPS-added medium induced the most CRM197 protein expression after 24 hours of induction, followed by Tris and phosphate buffer.

Example 3-A-2: pH value of expression Medium

The periplasmic expression of PelB-CRM197 was tested in media with various pH values of 1M MOPS, e.g. pH 7.0, 7.5 or 8.0. Strains No. 3-14-8 were inoculated and cultured in media of various pH values of 7.0, 7.5 or 8.0. The cultured bacteria were harvested at different time points, i.e. 3 and 24 hours, to examine the effect of pH on the degree of CRM197 expression. Harvested cells were disrupted with lysis buffer. Mu.g of total soluble protein was fractionated by 10% SDS-PAGE.

The results in figure 6 show that medium ph8.0 induced the most CRM197 protein expression after 24 hours of induction, followed by 7.5 and 7.0.

Example 4: analysis of the cleavage site at the N-terminus of pelBs-CRM 197

Since the soluble form of CRM197 was successfully expressed, a check was then made to see if the PelB signal sequence had been accurately and completely removed from the CRM197 recombinant protein by cellular proteinases. Thus, liquid chromatography-mass spectrometry (LC-MS) analysis was performed on partially purified CRM 197. In this experiment, two batches of CRM197 expressing cells were prepared: one batch was cultured in phoA medium and the other batch used MTB as basal medium in a fed-batch system (yeast extract 24 g/L; phytone peptone 12 g/L; glycerol 8 g/L; sodium chloride 5 g/L; potassium dihydrogen phosphate 0.232 g/L; dipotassium hydrogen phosphate 1.643 g/L). The harvested cells were then subjected to one-step DEAE chromatography to obtain homogenized CRM197 for LC-MS analysis. The results of LC-MS and CID analysis (fig. 7) clearly show that no PelB signal sequence was observed in these partially purified samples, and no N-terminal truncated form of CRM197 protein, indicating that the PelB signal sequence has been accurately and completely removed from the recombinant CRM 197.

Example 5: pelBs-CRM 197 disulfide bond formation assay

CRM197 contains four cystines and can form 2 internal disulfide bonds between Cys186-Cys201 and Cys461-Cys 471. Thus, the partially purified CRM197 described above was also used for disulfide bond analysis. The results in fig. 8 show that 2 disulfide bonds, Cys186-Cys201 and Cys461-Cys471, were identified in the two CRM197 samples, and no free thiol and disturbed disulfide bonds were identified. The results indicated that the recombinant CRM197 product formed the correct disulfide bonds.

Example 6: PelBs-CRM 197_ pEGm-phoA 1.0BL21#3-14-8 bacterial strain stability assay.

Protein expression and plasmid stability analyses were performed to assess the stability of CRM197 expressing strains, such as strains 3-14-8. To evaluate strain stability after several subcultures, the bacterial strains were continuously cultured and maintained at an exponential phase for up to 96 passages (doubling time of about 40 minutes in phoA medium, data not shown). Briefly, a vial of pelBs-CRM 197_ pEGm-phoA 1.0BL21 #was placed

3-14-8RCB was thawed and 0.4mL of glycerol stock was inoculated into 40mL of phoA medium (containing 30. mu.L/mL kanamycin) in a 250mL flask and cultured at 37 ℃ at 225 rpm. When OD is reached₆₀₀When 1 (about 4 hours) was reached, 0.4mL of culture was transferred to another flask with 40mL of fresh phoA medium. This process was repeated 16 times. Certain stages of cultured cells (passage 6, 66 and 96) were collected and glycerol stocks were also generated and stored at-75 ℃ for further analysis.

Example 6-1: protein expression stability analysis

In order to check CRM197 expression stability, CRM197 expression was performed on three different subcultured CRM197 strains No. 3-14-8. Protein expression methods as described previously, the final CRM197 expression analysis is shown in figure 9. The results clearly show that after 27.5 hours of culture in phoA medium, cells cultured on generations 6, 66 and 96 had comparable levels of CRM197 expression, as analyzed by 10% SDS-PAGE (FIG. 9 (A)). In addition, OD measured after 4 and 27.5 hours incubation₆₀₀The values are very similar (table 2): 4 hours 0.552-0.592 and 27.5 hours 3.402-3.576, indicating similar growth rates for these three generations of strains. Therefore, pelBs-CRM 197_ pEGm-phoA 1.0BL21#3-14-8RCB maintained good expression stability in 96 generations.

TABLE 2

Algebra	OD600(4 hours)	OD600(27.5 hours)
			6	0.576	3.576
66	0.592	3.402
			96	0.552	3.408

Example 6-2: plasmid stability analysis

To confirm whether the CRM197 expression vector also maintained its stability without deletion, replication or inversion of the DNA fragment, plasmids extracted from the 6 th, 66 th and 96 th generation cells were restriction enzyme digested and DNA sequenced. Two sets of restriction enzymes were used for plasmid digestion. HindIII and EcoRI release the pelBs-CRM 197 gene fragment (about 1.67kb) and the vector backbone (about 5.13 kb). On the other hand, EcoRV has 2 recognition sites in pelBs-CRM 197_ pEGm-phoA 1.0, one on the CRM197 gene and the other on the kanamycin gene, releasing 2 DNA fragments (about 2.65 and 4.15kb, respectively). The results of restriction enzyme mapping analysis on FIGS. 9(B) and 9(C) show that the expected DNA fragment was observed when 0.5. mu.g of plasmid from three generations of different cells was digested with HindIII/EcoRI or EcoRV and analyzed on a 1% agarose gel. In addition, DNA sequencing of plasmids extracted from 96 generations of cells was also performed from Genomics BioSci & Tech (Table 1, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13) and the correct sequence of the gene encoding CRM197 was confirmed (data not shown). In summary, the CRM197 expressing strains of the present application maintain stable plasmid stability after up to 96 passages of subculture.

Examples 6 to 3: plasmid copy number Retention analysis

In addition, the plasmid copy number retention rate after 96 passages was examined. For this purpose, methods based on quantitative-on-the-spot PCR (qPCR) were applied to determine the plasmid copy number in E.coli (Lee et al, 2006). To calculate absolute and relative plasmid copy numbers, the CRM197 gene in the plasmid was detected as the target gene using two sets of primers (Table 1, SEQ ID NO:14 and SEQ ID NO: 15). DXS gene (D-1-deoxyxylulose-5-phosphate synthase, D-1-deoxyxylulose5-phosphate synthase) in host chromosome was used as reference gene (Table 1, SEQ ID NO:16 and SEQ ID NO: 17). Because CRM197 and DXS are single copy genes in a vector and an Escherichia coli chromosome, the copy number of the plasmid can be determined as the ratio of CRM197 to DXS. To calculate the exact copy number of the plasmid (target gene) and the reference gene, 2 standard linear lines were first generated, converting the plasmid molecules into Ct values via known molecules (calculated from concentrations) of pelBss-CRM197_ pEGm phoA 1.0 and DXS _ pEGm phoA 1.0. Next, qPCR was performed on total DNA (containing pelBss-CRM197 plasmid and genomic DNA) extracted from the 6 th, 66 th and 96 th generation cells, and the amounts of CRM197 gene (plasmid) and DXS gene (genome) were calculated. Finally, absolute plasmid copy number was obtained by the amount of CRM 197/DXS. As shown in Table 3, the copy number retention was further calculated by dividing by the plasmid copy number of the 6 th generation sample. The results of 2 experiments showed that the plasmid copy number retention remained at least 80% up to passage 96.

TABLE 3

It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed embodiments. It is intended that the specification and examples be considered as exemplary only, with a true scope of the disclosure being indicated by the following claims and their equivalents.

Reference to the literature

The references listed below and cited herein are incorporated by reference into this specification, unless the specification expressly indicates otherwise.

1.Craig,S.P.III,Yuan,L.,Kuntz,D.A.,McKerrow,J.H.,and Wang,C.C.,1991.High level expression in Escherichia coli of soluble,enzymatically active schistosomal hypoxanthine/Guanine phosphoribosyltransferase and trypanosomal ornithine decarboxylase.Proc.Natl.Acda.Sci.USA.88,2500-2504.

2.Lee,C.,Kim,J.,Shin,S.G.and Hwang,S.,2006.Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli.J.Biotechnol.123,273-280.

Sequence listing

<110> Taiwan Haoyang technology GmbH

Liu, Lee-Cheng

<120> CRM197 protein expression

<130> 19P0065(19Q0057)

<150> US 62/619449

<151> 2018-01-19

<160> 17

<170> PatentIn version 3.5

<210> 1

<211> 1608

<212> DNA

<213> Artificial sequence

<220>

<223> CRM197 DNA sequence

<400> 1

ggtgcagatg atgttgttga tagcagcaaa agtttcgtga tggaaaactt cagcagctat 60

catggcacca aaccgggtta tgtggatagc attcagaaag gtattcagaa accgaaaagc 120

ggcacccagg gtaattatga tgatgattgg aaagagttct acagcaccga taacaaatat 180

gatgcagcag gttatagcgt ggataatgaa aatccgctga gcggtaaagc cggtggtgtt 240

gttaaagtta cctatccggg tctgaccaaa gttctggcac tgaaagttga taatgccgaa 300

accatcaaaa aagaactggg tctgagcctg accgaaccgc tgatggaaca ggttggcacc 360

gaagaattta tcaaacgttt tggtgatggt gcaagccgtg ttgtgctgag cctgccgttt 420

gcagaaggta gcagcagcgt tgaatatatc aataattggg aacaggcaaa agccctgagc 480

gttgaactgg aaatcaattt tgaaacccgt ggtaaacgtg gtcaggatgc aatgtatgaa 540

tacatggcac aggcatgtgc aggtaatcgt gttcgtcgta gcgttggtag cagcctgagc 600

tgtattaatc tggattggga tgtgattcgc gacaaaacca aaacgaaaat cgaaagcctg 660

aaagaacatg gtccgatcaa aaacaaaatg agcgaaagcc cgaataaaac cgtgagcgaa 720

gaaaaagcaa aacagtatct ggaagaattt caccagaccg cactggaaca tccggaactg 780

agcgaactga aaaccgttac cggcaccaat ccggtttttg ccggtgcaaa ttatgcagca 840

tgggcagtta atgttgcaca ggttattgat agcgaaaccg cagataatct ggaaaaaacc 900

accgcagcac tgagcattct gcctggtatt ggtagcgtta tgggtattgc agatggtgca 960

gtgcatcata ataccgaaga aattgttgcc cagagcattg ccctgagcag tctgatggtt 1020

gcccaggcaa ttccgctggt tggtgaactg gttgatattg gttttgcagc ctataacttt 1080

gtcgagagca tcattaacct gtttcaggtt gtgcataaca gctataatcg tccggcatat 1140

agtccgggtc ataaaaccca gccgtttctg catgatggtt atgcagttag ctggaatacc 1200

gttgaagata gcattattcg taccggcttt cagggtgaaa gcggtcatga tatcaaaatt 1260

accgcagaaa atacaccgct gccgattgcc ggtgttctgc tgccgaccat tccgggtaaa 1320

ctggatgtga ataaatccaa aacccacatt agcgtgaacg gtcgtaaaat tcgtatgcgt 1380

tgtcgtgcaa ttgatggtga tgttaccttt tgtcgtccga aaagtccggt ttatgttggt 1440

aatggtgttc atgcaaatct gcatgttgca tttcatcgta gctccagcga aaaaattcat 1500

agcaatgaaa ttagcagcga tagcattggt gttctgggtt atcagaaaac cgtggatcat 1560

accaaagtga atagcaaact gagcctgttc tttgaaatca aaagctaa 1608

<210> 2

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> PelB pectate lyase B) amino acid signal sequence

<400> 2

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala

20

<210> 3

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Kan-F

<400> 3

tgaaaaagga agagtatgag ccatattcaa cgg 33

<210> 4

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Kan-R

<400> 4

aacttggtct gacagttaga aaaactcatc gagcat 36

<210> 5

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> pTAC-F

<400> 5

ctgtcagacc aagtttactc atatatac 28

<210> 6

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> pTAC-R

<400> 6

actcttcctt tttcaatatt attgaa 26

<210> 7

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> phoA-pTAC-1-F

<400> 7

ctttgttttt attttttaat gtatttgtac ataggagata taatatgaag cttcctcg 58

<210> 8

<211> 59

<212> DNA

<213> Artificial sequence

<220>

<223> phoA-pTAC-1-R

<400> 8

cgactataag tctcggccgt gacaacttta tgacagaatt tcagaaggat cctctacgc 59

<210> 9

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> pelB-F

<400> 9

ctgctcctcg ctgcccagcc ggcgatggcc ggtgcagatg atgttgttga ta 52

<210> 10

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> pelB-R

<400> 10

cagaccagca gcagcggtcg gcagcaggta tttaagctta tgcggccttg a 51

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> phoA-SEQ-F

<400> 11

gcgtagagga tccttctgaa at 22

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> pTAC-SEQ-R

<400> 12

ctgtatcagg ctgaaaatct tctc 24

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> CRM197-433-F

<400> 13

gcagcagcgt tgaatatatc a 21

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> EG-197(II)-F

<400> 14

gcgaactgaa aaccgttacc 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> EG-197(II)-R

<400> 15

accaatacca ggcagaatgc 20

<210> 16

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> DXS-XhoI-F

<400> 16

actgctcgag taagttttga tattgccaaa tacccgacc 39

<210> 17

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> DXS-EcoRI-R

<400> 17

actggaattc ttatgccagc caggccttga 30

PCT/RO/134 Table