RU2803949C1 - Method for crm197 protein expression - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: nucleic acid construct for expression of the CRM197 protein is proposed, containing a nucleic acid encoding a signal sequence that contains the sequence SEQ ID NO: 6–7, 8–10 and SEQ ID NO: 8 and the CRM197 protein gene. An expression vector for expression of the CRM197 protein containing the specified nucleic acid construct, a recombinant microorganism for the expression of the CRM197 protein containing the specified nucleic acid construct or the specified expression vector, as well as a method for producing the CRM197 protein are also proposed.

EFFECT: invention ensures efficient expression of CRM197 protein and efficient secretion of CRM197 protein into the periplasm without changing the pH of the medium.

9 cl, 18 dwg, 8 tbl, 5 ex

Description

Field of technology

[1] The present invention relates to a signal sequence for expression of CRM197 protein in E. coli and secretion of CRM197 protein into the periplasm and its use, and more particularly to a signal sequence for expression of CRM197 protein, a nucleic acid encoding said signal sequence, nucleic acid constructs or an expression vector containing a nucleic acid and a CRM197 protein gene, a recombinant microorganism into which the nucleic acid construct or expression vector is introduced, and a method for producing the CRM197 protein, including cultivating the recombinant microorganism.

[2]

State of the art

[3] Diphtheria toxin (DT) is a protein exotoxin synthesized and secreted by the pathogenic strain Corynebacterium diphtheriae. Diphtheria toxin is an ADP-ribosylating enzyme that is secreted as a 535-residue proenzyme that is divided into two fragments (fragments A and B) by treatment with a trypsin-like protease. Fragment A is the catalytically active region and is an NAD-dependent ADP-ribosyltransferase that specifically targets the protein synthesis factor EF-2, thereby inactivating EF-2 and interrupting protein synthesis in cells.

[4] CRM197 was discovered by isolating various non-toxic forms and partially toxic immunologically cross-reactive forms (CRMs or cross-reactive substances) of diphtheria toxin (Uchida et al., Journal of Biological Chemistry 248, 3845-3850, 1973). Preferably, the CRM may be of a predetermined size and composition, including all or part of the DT.

[5] CRM197 is an enzymatically highly inactive and nontoxic form of diphtheria toxin that contains a single amino acid substitution (G52E). This mutation imparts intrinsic flexibility to the active site loop located upstream of the NAD binding site, thereby reducing the binding affinity of CRM197 to NAD and eliminating DT toxicity (Malito et al., Proc. Natl. Acad. Sci. USA 109 (14): 5229-342012 ). CRM197, like DT, has two disulfide bonds. One disulfide bond links Cys186 to Cys201, thereby linking fragment A to fragment B. Another disulfide bond links Cys461 to Cys471 in fragment B. DT and CRM197 have nuclease activity acquired from fragment A (Bruce et al., Proc. Natl. Acad Sci. USA 87, 2995-8, 1990).

[6] A number of antigens have low immunogenicity, especially in infants and young children, unless they are chemically bound to proteins and thus formulated as conjugates or conjugate vaccines. In these conjugate vaccines, the protein component is also called the “carrier protein.” CRM197 is commonly used as a carrier protein in protein-carbohydrate conjugation and hapten-protein conjugation. CRM197, as a carrier protein, has several advantages over diphtheria toxoid as well as other toxoid proteins (Shinefield Vaccine, 28: 4335, 2010).

[7] Methods for producing diphtheria toxin (DT) are well known in the art. For example, DT can be produced by purifying the toxin from a culture of Corynebacterium diphtheriae followed by chemical detoxification, or by purifying a recombinant or genetically detoxified analogue of the toxin.

[8] The abundance of proteins made it impossible to mass produce diphtheria toxins such as CRM197 for use in vaccines. This problem was previously addressed by expressing CRM197 in E. coli (Bishai, et al., J. Bacteriol. 169: 5140-5151), and Bishai et al. reported a recombinant fusion protein containing a toxin (including the toxin signal sequence) resulting in the production of degraded proteins.

[9] Compared to cytoplasmic production, the production of bacterial toxins in the periplasm is characterized by the fact that i) the protein is produced in a mature form after cleavage of the signal peptide, or ii) the E. coli periplasm is an oxidizing environment, which allows the formation of disulfide bonds, which can promote production of soluble, correctly folded proteins, iii) the periplasm of E. coli contains less protease than the cytoplasm, which helps avoid proteolytic degradation of the expressed proteins, and iv) the periplasm also contains a smaller amount of proteins, which allows for the production of recombinant protein of higher purity.

[10] In general, the presence of signal sequences on proteins facilitates the transport (prokaryotic hosts) or secretion (eukaryotic hosts) of proteins into the periplasm. In prokaryotic hosts, signal sequences coordinate newly formed proteins with the periplasm across the inner membrane and are then cleaved. That is, the search for signal sequences that can more efficiently mass produce commercially needed proteins is important, and it is necessary to develop recombinant microorganisms.

[eleven]

[12] Accordingly, as a result of significant efforts to develop a method for producing the CRM197 protein in an efficient and economical manner, the present inventors selected specific signal sequences, designed nucleotide sequences, combining codon context with secondary structure, in order to optimize translation in E. coli, optimize the expression in E. coli of CRM197 nucleotide sequences encoding the CRM197 protein, and found that CRM197 is efficiently expressed in E. coli and efficiently secreted into the periplasm without changing the pH when used, thereby completing the present invention.

[13]

[14] The information disclosed in this “Background of the Art” section is provided only to better understand the background of the present invention, and therefore it may not include information constituting prior art that is already apparent to those skilled in the art.

[15]

Brief description of the invention

[16] One of the objects of the present invention is to provide a signal sequence for the expression of a CRM197 protein having a specific sequence, a nucleic acid encoding the signal sequence, and a method for producing the CRM197 protein using the nucleic acid to maximize the expression of the CRM197 protein while minimizing the toxicity of the CRM197 protein to E. coli by secretion of the CRM197 protein into the E. coli periplasm.

[17]

[18] To achieve the objects, the present invention provides a signal sequence for expression of the CRM197 protein represented by any of the amino acid sequences of SEQ ID NO: 13 to SEQ ID NO: 21.

[19] The present invention also provides a nucleic acid encoding a signal sequence for expression of the CRM197 protein.

[20] The present invention also relates to a nucleic acid construct or expression vector containing a nucleic acid and a CRM197 protein gene, a recombinant microorganism into which the nucleic acid construct or expression vector is introduced, and a method for producing CRM197 protein, including cultivating the recombinant microorganism.

[21]

Brief description of graphic materials

[22] In FIG. 1 is a schematic diagram illustrating the TPB1TV1.3 expression module, where Ptrc denotes the trc promoter, RBS denotes the A/U-rich enhancer + SD, λtR2 and T7Te, and rrnB T ₁ T ₂ denotes transcription terminators, and Bsal and Dral denote restriction enzyme sites , used for cloning.

[23] FIG. 2 illustrates the E. coli expression plasmid pHex1.3, where Ori (pBR322) denotes the pBR322 origin of replication, KanR denotes the kanamycin marker, lacI denotes the lacI gene, and other symbols are the same as in FIG. 1.

[24] In FIG. 3 is a schematic diagram illustrating the preparation of the CRM197 expression plasmid, where T1 denotes the transcription terminator λtR2 and T7Te, T2 denotes the transcription terminator rrnB T ₁ T ₂ , arrows denote PCR primers, letters A, B, C and D denote homologous regions for LIC , and the remaining symbols are the same as in FIG. 1.

[25] FIG. 4 illustrates the expression behavior of the CRM 197 fusion with L3 and L5 in various E. coli strains, as well as the results of Coomassie staining (left) and Western blotting (right) after SDS-PAGE of all E. coli cells, where C is C2894H, B is BL21 (DE3), W is W3110-1, O is Origami™ 2, S is shuffle, Cv is C2984H containing pHex1.3 used as a negative control, L3 and L5 are signal sequence fusions, and CRM197 is the CRM197 reference and cells are loaded into each well at a density corresponding to an _OD600 of 0.025 for Coomassie staining and at a density corresponding to an _OD600 of 0.0005 for Western blotting.

[26] FIG. 5 illustrates the effect of inducer type and concentration on L5-induced CRM197 expression and illustrates Coomassie staining (left) and Western blot (right) cases after SDS-PAGE, where the numbers of loaded cells are the same as in FIG. 4, I indicates the insoluble fraction, S indicates the soluble fraction, (A) shows the culture at 25°C, and (B) shows the culture at 30°C.

[27] In FIG. Figure 6 shows the position of the CRM197 protein induced by fusion with L5, where Coomassie staining after SDS-PAGE is shown at the top, Western blotting is shown at the bottom, Cr represents the reference CRM197, the arrow shows the position of mature CRM197, P1 indicates the supernatant obtained after treatment with plasmatic induction buffer membranes, P2 denotes the periplasmic fraction, and Cy denotes the cytoplasmic fraction.

[28] FIG. 7 illustrates the effect of inducer type and concentration on L3-induced CRM197 expression, where (A) shows a culture at 25°C and (B) shows a culture at 30°C.

[2 9] In FIG. Figure 8 shows the position of the CRM197 protein induced by fusion with L3.

[30] In FIG. 9 shows the changes in strain L3 pH, temperature, paddle speed and dissolved oxygen (DO), as well as the time of addition of the expression inducer, where (A) shows the case when the temperature is maintained at 30°C, and (B) shows the case when Before induction of expression, the temperature is reduced to 25°C.

[31] In FIG. Figure 10 shows changes in strain L5 pH, temperature, paddle speed, and dissolved oxygen (DO), as well as the time of addition of the expression inducer.

[32] In FIG. 11 shows the change in CRM197 protein expression before and after the addition of an expression inducer during culture, where (A) shows expression of strain L3 induced at 30°C, (B) shows expression of strain L3 induced at 25°C, (C) shows expression strain L5, induced at 25°C. 200 ng of reference CRM197 was loaded into lines 3 and 10 in (A), lines 4 and 10 in (B), and lines 3 and 9 in (C). Lines 1 and 2 in (A), lines 1, 2 and 3 in (B) and lines 1 and 2 in (C) represent cell culture solutions before addition of the expression inducer, and line 16 in (A) and line 16 in ( B) are supernatants after the end of cultivation, subsequent lines are cell culture solutions taken every 2 hours after adding the inducer. The loading amount is the same as in FIG. 4.

[33] FIG. 12 illustrates the expression and partitioning behavior of the CRM197 protein in L3/L5 strains, where (A) illustrates the behavior of the protein when partitioned in culture using L3, (B) illustrates the behavior of the protein when partitioned in culture using L5, line 1 was loaded with 200 ng of reference CRM197, line 2 is the total cell culture solution after completion of culture, line 3 is the supernatant after treatment with plasma membrane induction buffer, lines 4 and 7 are periplasmic fractions (line 7 contains 4 times more protein than line 4), and lines 5 and 6 represent the cytoplasmic fractions.

[34] FIG. 13 illustrates the result of SDS-PAGE of samples purified by DEAE chromatography (A) and HA chromatography (B). In (A), line 1 denotes CRM 197 produced by Corynebacterium, line 2 denotes the protein present in the isolated periplasmic fraction, line 3 denotes the sample before loading into DEAE chromatography, which is concentrated twice after ultrafiltration, lines 4 and 5 are used to detect contaminant proteins , with the exception of CRM197, using flow chromatography with DEAE and wash solutions, line 6 is an eluted sample from which impurities have been removed, line 7 is a sample eluted using high-concentration salt to remove all resin-bound proteins. The HA Elu in (B) is CRM197 eluted after removal of protein not bound to the HA resin.

[35] FIG. 14 illustrates the result of SEC-HPLC analysis of the final purified CRM197 where the purity was 99% or more.

[36] In FIG. Figure 15 shows the results of SDS-PAGE (left) and Western blotting (right). Line 1 is CRM197 produced by Corynebacterium and line 2 is CRM 197 produced by E. coli (L3). Both CRM 197s showed stripes in the same position.

[37] FIG. 16 illustrates the result of an intact protein molecular weight analysis using LC/MS, where the molecular weight is 58409 Da, which is the theoretical molecular weight.

[38] FIG. 17 illustrates the result of circular dichroism (CD) analysis and indicates that there is no difference in higher order structure between CRM197 ( ^• ) produced by Corynebacterium and CRM197 ( ^X ) produced by E. coli pHex-L3.

[39] FIG. 18 illustrates the result of fluorescence spectrum analysis where CRM197 ( ^• ) produced by Corynebacterium and CRM197 (solid line) produced by E. coli pHex-L3 had the same maximum emission wavelength of 338 nm.

[40]

Detailed Description and Preferred Embodiments of the Invention

[41] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as understood by those skilled in the art to which the present invention relates. In general, the nomenclature used herein is well known in the art and is commonly used.

[42]

[43] In one embodiment of the present invention, 9 signal sequences are fused to the CRM197 protein to induce its expression. For each signal sequence, nucleotide sequences (SEQ ID NO: 4 - SEQ ID NO: 12) optimized for translation were designed, taking into account the codon context and secondary structure of the mRNA. These constructs were inserted into the expression plasmid pHex1.3, and CRM197 expression was observed in five E. coli strains to find the optimal E. coli strain for each construct. In addition, the culture temperature of the selected E. coli was determined, as well as the type and concentration of the inducer. As a result of transformation of various E. coli strains with constructs, the CRM197 protein could be expressed in a soluble form even in strains in which genes (trxB, gor) associated with redox potential were not constructed, and could be secreted in the periplasm.

[44]

[45] Thus, in one aspect, the present invention is directed to a signal sequence for expression of the CRM197 protein represented by any of the amino acid sequences of SEQ ID NO: 13 through SEQ ID NO: 21.

[46] In another aspect, the present invention is directed to a nucleic acid encoding a signal sequence for expression of the CRM197 protein.

[47] In the present invention, the nucleic acid may be represented by any of the nucleotide sequences of SEQ ID NO: 4 to SEQ ID NO: 12, preferably represented by the nucleotide sequences of SEQ ID NO: 6 or SEQ ID NO: 8, but is not limited to it.

[48] As used herein, the term “signal sequence for expression of CRM197 protein” means a signal sequence for expression of CRM197 protein and secretion of CRM197 protein into the periplasm.

[49] In one embodiment of the present invention, a signal sequence of a protein targeting the outer membrane of E. coli and a signal sequence derived from phage M13 (Table 3) were selected to secrete the CRM197 protein into the periplasm. Nucleotide sequences (SEQ ID NO: 4 through SEQ ID NO: 12) were designed by combining codon context with secondary structure to optimize translation of the selected signal sequence in E. coli.

[50]

[51] In another aspect, the present invention is directed to a nucleic acid construct comprising a nucleic acid encoding a signal sequence for expression of a CRM197 protein and a CRM197 protein gene.

[52] In another aspect, the present invention provides an expression vector comprising a nucleic acid encoding a signal sequence for expression of the CRM197 protein and a CRM197 protein gene.

[53] In one embodiment of the present invention, the DNA sequence encoding the amino acid sequence of the CRM197 protein (SEQ ID NO: 3) has also been optimized for expression of E. coli (SEQ ID NO: 2). The DNA fragment of each designed signal sequence and the optimized DNA fragment of CRM197 were inserted into the pHex1.3 plasmid (FIG. 3).

[54] In the present invention, any CRM197 protein gene can be used without limitation as long as it is a gene encoding a CRM197 protein. The CRM197 protein gene may preferably be represented by the nucleotide sequence of SEQ ID NO: 2, but is not limited to this.

[55] The term “transformation” as used in this application means the introduction into cells of a specific foreign DNA chain from the outside. The host microorganism containing the introduced DNA strand is called a "transformed microorganism". As used herein, the term “transformation,” meaning the introduction of DNA into a host and rendering the DNA replicable by an extrachromosomal factor or chromosomal integration, indicates that a vector containing a polynucleotide encoding a target protein is introduced into a host cell, or a polynucleotide encoding a target protein , are integrated into the host cell chromosome to express the protein encoded by the polynucleotide in the host cell. The transformed polynucleotide includes both a transformed polynucleotide inserted into and located within the chromosome of a host cell, and a transformed polynucleotide located outside the chromosome, provided that it can be expressed in the host cell.

[56] As used herein, the term “nucleic acid construct” includes both a nucleic acid construct inserted into and located within the chromosome of a host cell, and a nucleic acid construct located outside of the chromosome, provided that it can be expressed on the chromosome of the cell -the owner.

[57] Additionally, as used herein, the term “polynucleotide” is used interchangeably with the term “nucleic acid” and includes DNA and RNA encoding the target protein. The polynucleotide can be introduced in any form, provided that it can be introduced into and expressed in a host cell. For example, the polynucleotide can be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for self-expression. An expression cassette typically contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal that is operably linked to a nucleic acid. The expression cassette may contain an expression vector that allows for self-replication. The polynucleotide may also be introduced into a host cell in its native form and operably linked to a sequence required for expression in the host cell.

[58] As used herein, the term “vector” means a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. Vectors can be plasmids, phage particles, or simple potential genomic inserts. When transformed into a suitable host, the vectors may replicate or perform functions independent of the host genomes, or some of them may be integrated with the genomes. Plasmids are currently the most commonly used form of vector. Thus, the terms "plasmid" and "vector" are used interchangeably.

[59] Considering the purposes of the present invention, the use of a plasmid vector is preferred. Typical plasmid vectors that can be used to achieve objectives include (a) an origin of replication to efficiently carry out replication to include several or several hundred plasmid vectors in each host cell, (b) an antibiotic resistance gene for host cell screening, transformed with plasmid vectors and (c) a restriction enzyme cleavage site into which a fragment of foreign DNA is inserted. Even if a corresponding restriction enzyme cleavage site is missing, the vector and foreign DNA can be easily ligated using a synthetic oligonucleotide adapter or linker according to a conventional method.

[60] Additionally, when a gene is aligned with another nucleic acid sequence based on the functional relationship between them, it is said to be “functionally linked” to it. These may be gene(s) and regulatory sequence(s) linked in such a way as to allow expression of the gene when a suitable molecule (eg, a transcription activator protein) is linked to the regulatory sequence(s). For example, the DNA of a presequence or secretory leader is operably linked to the DNA of a polypeptide when expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence when it influences transcription of the sequence; a ribosome binding site is operably linked to a coding sequence when it affects transcription of the sequence; or the ribosome binding site is operably linked to the coding sequence when positioned to facilitate translation.

[61] Generally, the term "operably linked" means that the linked DNA sequence is in contact or that the secretory leader is in contact with it and is present in frame. However, the enhancer should not come into contact with them. Linking of these sequences is accomplished by ligation at convenient restriction enzyme sites. When such a site does not exist, a synthetic oligonucleotide adapter or linker is used in accordance with the usual method.

[62] In the present invention, the expression vector may further comprise a Trc promoter.

[63] In the present invention, the expression vector may be pHex1.3, but is not limited to it.

[64] CRM197 is known to be highly toxic to E. coli due to its nuclease activity. Thus, expression of CRM197 under undesirable conditions may negatively affect the growth of E. coli. The E. coli expression plasmid pHex1.3 contains the Lacl gene and can suppress the background expression of the trc promoter, and the TPB1Tv1.3 expression module built into this plasmid (FIG. 1) is designed to suppress the expression of CRM197, transcribed from the promoter obtained from the plasmid, by inserting transcription terminator tR2, obtained from phage λ, and transcription terminator Te, obtained from phage T7, into the upstream part of the expressed CRM197 gene, and transcription terminator rrnB T1T2, obtained from E. coli, into its downstream part.

[65]

[66] In another aspect, the present invention is directed to a recombinant microorganism into which a nucleic acid construct or expression vector has been introduced.

[67] In the present invention, the recombinant microorganism may be Escherichia coli, but is not limited to it.

[68] As the recombinant microorganism, host cells having high DNA introduction efficiency and high expression efficiency of the introduced DNA are usually used, all bacteria, yeast, mold, etc., that is, all microorganisms including prokaryotic and eukaryotic cells are available, and in the example of the present invention, E. coli was used, but the present invention is not limited to them, and any type of microorganism can be used as long as the CRM197 protein can be expressed sufficiently.

[69] It should be understood that not all vectors function equally in expressing the DNA sequences of the present invention. Likewise, not all hosts function equally for the same expression system. However, those skilled in the art will be able to make appropriate selections among various vectors, expression control sequences, and hosts without undue experimental burden and without departing from the scope of the present invention. For example, vector selection should be based on the host, since the vector must replicate within it. The number of vector replicates, the ability to control the number of vector replicates, and the expression of other proteins encoded by the corresponding vector, such as the expression of antibiotic markers, should also be considered.

[70] The transformed recombinant microorganism can be obtained by any known transformation method.

[71] In the present invention, the method of inserting a gene into the chromosome of host cells can be selected from well-known methods of genetic manipulation, for example, methods using retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes simplex virus vectors, poxviral vectors, lentiviral vectors or non-viral vectors.

[72] Alternatively, transformation can be accomplished by direct insertion of a nucleic acid construct into the host cell chromosome in addition to the use of expression vectors.

[73] In general, electroporation, lipofection, ballistic delivery, virosomes, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virions, chemically stimulated DNA influx, calcium phosphate (CaPO ₄ ) precipitation can be used. calcium chloride ( _CaCl2 ) precipitation, microinjection, lithium acetate-DMSO method, etc.

[74] Sonoporation, such as methods using the Sonitron 2000 system (Rich-Mar), can also be used to deliver nucleic acids, and other representative nucleic acid delivery systems include Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Maryland) and BTX Molecular System (Holliston, Mass.). Methods for lipofection are disclosed in US Pat. No. 5,049,386, US Pat. No. 4,946,787, and US Pat. No. 4,897,355, and lipofection reagents are commercially available, such as TRANSFECTAM™ and LIPOFECTIN™. Cationic or neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include Felgner lipids (WO 91/17424 and WO 91/16024), which can be delivered to cells via ex-vivo transduction and to target tissues via in-vivo transduction. Methods for preparing a lipid:nucleic acid complex containing a target liposome, such as an immunolipid complex, are well known in the art (Crystal, Science., 270: 404-410, 1995; Blaese et al., Cancer Gene Ther., 2: 291 -297, 1995; Behr et al., Bioconjugate Chem., 5: 382389, 1994; Remy et al., Bioconjugate Chem., 5: 647-654, 1994; Gao et al., Gene Therapy., 2: 710- 722, 1995; Ahmad et al., Cancer Res., 52: 4817-4820, 1992; US Patent No. 4186183; US Patent No. 4217344; US Patent No. 4235871; US Patent No. 4261975; US Patent No. 4485054; US Patent No. 4501728 , US Patent No. 4774085, US Patent No. 4837028, US Patent No. 4946787).

[75] In one embodiment of the present invention, by transforming various E. coli strains with the resulting plasmid vector (Table 4), for L3 fusion, CRM197 was expressed in all strains, and for L5 fusion, CRM197 was expressed in strains except Origami™ 2 (FIG. 4). The L3 and L5 hybrids were able to express the CRM197 protein in a soluble form even in strains in which the redox-related genes (trxB, gor) were not engineered and the protein had the same physicochemical/immunological properties as protein isolated from parental strains. In addition, when fused to L3, the CRM197 protein can be expressed in a soluble form at both 25°C and 30°C (FIGS. 7 and 12A).

[76] A previous report showed that when cultured at pH 6.5 to 6.8 and then changed to pH 7.5 during induction, secretion of CRM197 into the periplasm was improved. In contrast, the strain prepared according to the present invention was found to efficiently secrete CRM197 into the periplasm without changing the pH of the medium (FIG. 12). In the case of fusion with L5 in a highly concentrated culture without a pH shift, the productivity of CRM197 was 3.7 g/L, and 2 g/L or more of CRM197 was secreted into the periplasm.

[77]

[78] In another aspect, the present invention provides a method for producing CRM197 protein, comprising (a) cultivating a recombinant microorganism into which a nucleic acid construct or expression vector has been introduced to produce CRM197 protein, and (b) isolating the resulting CRM197 protein.

[79] In the present invention, step (b) may include isolating the CRM197 protein secreted into the periplasm.

[80]

[81] Hereinafter, the present invention will be described in more detail with reference to examples. However, it will be apparent to those skilled in the art that these examples are presented only to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[82]

[83] Example 1: Preparation of CRM197 Overexpression Plasmid

[84] Example 1.1: Construction of plasmid pHex1.3

[85] The expression plasmid of E. coli pHex1.3 was obtained as follows. Following double cleavage of ptrc99a (Amann et al., Gene. 69, 301-15, 1988) Sspl and Dral, an approximately 3.2 kb DNA fragment is formed. purified by agarose electrophoresis. The kanamycin resistance gene was amplified by PCR. The template used herein was plasmid pCR2.1, and the primers used herein were KF2 and KR (Table 1).

[86]

[88]

[89] The PCR reaction solution was prepared using 2.5 mM of each dNTP, 10 pmol of each primer, 200 to 500 ng of template DNA, 1.25 U of PrimeSTAR HS DNA polymerase (Takara Bio Inc., Japan) and 50 μl reaction volume, and PCR was performed for 30 cycles, each of which included three steps at 98°C for 10 seconds, 60°C for 5 seconds, and 72°C min/kb. After double cleavage of the approximately 0.8 kb DNA fragment obtained by PCR using SamHI and HindIII, the DNA fragment was complemented with a Klenow fragment to form a blunt end and then ligated with the 3.2 kb DNA fragment. .n. obtained in the previous process using T4 DNA ligase. This reaction solution was transformed into E. coli C2984H to produce pHex1.1, in which the selection marker was changed from Amp to Km.

[90] The expression module TPB1Bv1.3, including promoters, RBS and transcription terminators (FIG. 1), was synthesized in Bioneer (SEQ ID NO: 1). The process of loading the TPB1Bv1.3 expression module on pHex1.3 is as follows. A DNA fragment (1255 bp) obtained by PCR amplification using the TPB1Bv1.3 expression module as a template and TPB_F and TPB_R as primers (Table 1) was assembled in vitro with a DNA fragment (4561 bp .), obtained by PCR amplification using pHex1.1. as template and pHex_F and pHex_R as primers by ligation-independent cloning (LIC; Jeong et al., Appl Environ Microbiol. 78, 5440-3, 2012) under the conditions shown in Table 2, and the resulting product was transformed into E. coli C2984H to obtain pHex1.3 (FIG. 2).

[91]

[93]

[94] LIC reaction conditions: Vectors were digested with restriction enzymes or linear DNA fragments were obtained by PCR. Inserts were prepared by PCR. The vectors were mixed with the inserts as shown in the table above and then allowed to react at room temperature for 2 minutes and 30 seconds.

[95]

[96] Example 1.2: Construction of the CRM197 gene

[97] The nucleotide sequence (CRM197ec) of CRM197 optimally expressed in E. coli was synthesized in GenScript (SEQ ID NO: 2). The amino acid sequence encoded by CRM197ec is SEQ ID NO: 3.

[98]

[99] Example 1.3: Signal sequence gene construction

[100] The signal sequences used for secretion of the CRM197 protein into the periplasm of E. coli are shown in Table 3 below (SEQ ID NO: 13-21). To optimize expression in E. coli, DNA with SEQ ID NO: 4-12 was synthesized taking into account codon context and secondary structure (Table 3).

[101]

[103]

[104] Example 1.4: Construction of a plasmid for overexpression of CRM197

[105] Plasmids for overexpression of CRM 197 in E. coli and secretion of CRM 197 into the periplasm were prepared as shown in FIG. 3. After double digestion of pHex1.3 with BsaI and DraI, a DNA fragment of approximately 5 kb was isolated by agarose electrophoresis. The L1-L9 signal sequences were amplified by PCR using the primers and templates shown in Table 1. The CRM197 DNA fragment for fusion of the CRM197 gene with each signal sequence using the LIC method was amplified by PCR using the primers and templates shown in Table 1. pHex1.3, digested with BsaI and DraI, each signal sequence fragment and its compatible CRM197 fragment were assembled in vitro using the LIC conditions described above, and then the resulting product was transformed into E. coli C2984H to obtain plasmids pHex-L1-CRM, pHex- L2-CRM, pHex-L3-CRM, pHex-L4-CRM, pHex-L5-CRM, pHex-L6-CRM, pHex-L7-CRM, pHex-L8-CRM and pHex-L9-CRM containing CRM197, fused with the corresponding signal sequences.

[106]

[107] Example 2: Expression of CRM197 protein in E. coli

[108] pHex-L3-CRM and pHex-L5-CRM were selected from the plasmids obtained in Example 1, taking into account the level of expression and the degree of cell growth. After transforming pHex-L3-CRM and pHex-L5-CRM into the E. coli strain of Table 4 below, the expression and expression position of CRM197 protein were evaluated.

[109]

[111]

[112] The cultivation method is as follows. The colony obtained on solid medium (10 g/l soyton, 5 g/l yeast extract, 10 g/l NaCl, 15 g/l agar) was cultured with shaking in liquid LB medium containing 100 mM potassium phosphate (pH 7, 5), km 50 μg/ml and 0.2% lactose, all cells were subjected to SDS-PAGE, and then CRM197 expression was analyzed using Coomassie staining and Western blotting (FIG. 4). For the L3 fusion, CRM197 protein was expressed in all strains used, while for the L5 fusion, growth was not possible in liquid medium when transformed into Origami™ 2. CRM197 protein expression was observed in other strains.

[113]

[114] Example 2.1: Expression of CRM197 by L5

[115] BL21 (DE3) containing pHex-L5-CRM was cultured in liquid LB medium (50 mL/500 mL baffled flask) containing 100 mM potassium phosphate (pH 7.5) and 50 μg/mL km until OD ₆₀₀ did not reach 0.4-0.6. Then, 0.2, 0.4, or 0.6% lactose or 0.02, 0.2, or 2 mM IPTG (isopropyl-β-D-1-thiogalactopianoside) was added as an inducer to induce expression. The culture temperature was 25°C or 30°C. After culture, the cells were removed, suspended in 50 mM potassium phosphate (pH 7.0) and then disrupted by ultrasound. After destruction, centrifugation was performed to separate the supernatant (soluble fraction) from the sediment (insoluble fraction). After SDS-PAGE of each sample, CRM197 protein expression was analyzed using Coomassie staining and Western blotting (FIG. 5). When an inducer (lactose, IPTG) was added and then cultured at 25°C, most of the expressed CRM197 protein was present in soluble form and had the same molecular weight as the reference CRM197, indicating that the expressed protein was mature CRM197. from which the L5 signal sequence was removed (FIG. 5A). On the other hand, when lactose was used as an inducer and culture was performed at 30°C, approximately 50% of the CRM197 protein was found in the insoluble fraction, and when IPTG was added as an inducer, most of the CRM197 protein was found in the insoluble fraction (FIG. 5B ).

[116] The periplasmic fraction was extracted using osmotic shock to determine the location where CRM197 was expressed. The process is as follows. BL21 (DE3) containing pHex-L5-CRM was cultured at 25°C and then centrifuged to isolate cells. Cells were resuspended in plasma membrane induction buffer [30 mM Tris-HCI (pH 8.0), 20% sucrose, 1 or 10 mM EDTA, 1 mM PMSF (phenylmethylsulfonyl fluoride)] until the cell concentration reached OD ₆₀₀ . equal to 10, and stirred at room temperature for 0.5 to 1 hour. Cells were then collected by centrifugation at 4000 × g for 15 minutes and the same amount of 30 mM cold (4°C or lower) Tris-HCI (pH 8.0) was added, followed by stirring at room temperature for 0.5-1 hour . The cells were then centrifuged at 4000 g for 15 minutes to obtain the supernatant (periplasmic fraction, P2). After treatment with plasma membrane induction buffer, the supernatant (P1), periplasmic fraction (P2) and cytoplasmic fraction were developed by SDS-PAGE, and then the expression of CRM197 and the position of expressed CRM197 was assessed using Coomassie staining and Western blotting (FIG. 5). It was found that L5 could successfully secrete CRM197 into the periplasm (FIG. 6). EDTA as a buffer for plasma membrane induction was more effective at 10 mM than at 1 mM.

[117]

[118] Example 2.2: Expression of CRM197 by L3

[119] BL21 (DE3) containing pHex-L3-CRM was expressed under the same conditions as in example 2.1. It was found that CRM197 induced by L3, unlike L5, was present in soluble form under all conditions (FIG. 7) and secreted into the periplasm (FIG. 8).

[120]

[121] Example 3: Culture of E. coli BL21 (DE3)

[122] Strain BL21 (DE3) containing pHex-L3-CRM or pHex-L5-CRM was cultured using the following method. During the main culture, feeding was carried out using the pH stat method, and the pH was maintained at 7.3 using a nutrient solution (600 g/L glucose, 30 g/L yeast extract) and alkali solution (14-15% ammonia). The compositions of solutions and media used for cultivation are shown in Tables 5 and 6.

[123]

[125]

[125]

[127]

[128] Single colony formed on modified LB agar medium [modified Luria-Bertani (LB) agar: 10 g/L soyton, 5 g/L yeast extract, 10 g/L sodium chloride, 15 g/L agar, 50 mg /l kanamycin] were inoculated into the seed culture medium, followed by incubation at 30°C for 18 hours. The resulting seed culture was again inoculated at a ratio of 1% (v/v) into the main culture medium (fermenter 3 L/5 L) and cultured at 30°C. The basic nutrient medium was prepared by adding 0.1% sterilized defoamer to the inoculum nutrient medium. After the optical density of the culture solution reached 30-40, the temperature was reduced to 25°C. Then 10 mM IPTG was added and cultivation was stopped after the absorbance of the culture solution reached 100-120.

[12 9] The behavior of an E. coli BL21 (DE3) culture containing pHex-L3-CRM in a 5 L fermenter is shown in FIG. 9, and the behavior of an E. coli BL21 (DE3) culture containing pHex-L5-CRM is shown in FIG. 10. The expression pattern of CRM197 during fermentation in a 5 L fermenter is shown in FIG. 11, and the expression yield was 1.1 to 1.2 g/L for the L3 fusion and 3.0 to 3.7 g/L for the L5 fusion. The amount of CRM197 was measured by comparing the relative amount with reference CRM197 using a densitometer (GS-900™, Bio-Rad Laboratories Ins., Hercules, California) after SDS-PAGE/Coomassie staining.

[130]

[131] Example 4: Protein Purification

[132] Example 4.1: Preparation of the periplasmic fraction from cell culture

[133] The procedure for isolating the periplasmic fraction from cells cultured in a 5-liter fermenter is as follows. The cell culture medium was centrifuged at 4°C and 4000 × g for 15 minutes to pellet the cells. The cell pellet was resuspended in plasma membrane induction buffer (Table 7) with modified protein based on an absorbance of 100 and stirred at room temperature for 0.5 to 1 hour.

[134]

[136]

[137] Cells were then collected by centrifugation at 4,000 × g for 15 minutes and the same amount of 30 mM cold (4°C or lower) Tris-HCI (pH 8.0) was added, followed by stirring at room temperature for 0.5 -1 hour. The cells were then centrifuged at 4000 g for 15 minutes to obtain the supernatant, and contaminants were removed using MF. SDS-PAGE analysis of the periplasmic fraction recovered from E, coli BL21 (DE3) containing pHex-L3-CRM by the process described above is shown in FIG. 12 (A), and SDS-PAGE analysis of the periplasmic fraction extracted from E. coli BL21 (DE3) containing pHex-L5-CRM is shown in FIG. 12(B). The amount of CRM197 protein present in the periplasmic fraction was found to be 1.2 g/L for strain L3 and 2.3 g/L for strain L5 (Table 8).

[138]

[140]

[141] Example 4.2: Purification of CRM197 protein

[142] The periplasmic fraction of pHex-L3-CRM culture medium was concentrated twice using a 10 kDa cutoff membrane using a TFF system and ultrafiltrated using ten volumes of 10 mM sodium phosphate solvent (pH 7.2). Purification was carried out using two-column processes using the AKTA pure system (GE Healthcare). The first column process was anion exchange chromatography (fast flow diethylaminoethyl sepharose resin, DEAE) and was used to remove nucleic acids and contaminating proteins. DEAE resin is negatively charged (-) and bound to a positively charged (+) protein. Unbound proteins and impurities were firstly extracted and removed from the ultrafiltered sample by DEAE chromatography, and then crude proteins with low binding capacity, except CRM197, were removed by a subsequent washing process depending on the salt concentration. Then, with increasing salt concentration, only CRM197 was eluted. SDS-PAGE analysis of the sample was performed during the purification process using DEAE chromatography, and the results are shown in FIG. 13(A). First, unbound impurities and proteins were first flow-removed from the DEAE-chromatography sample using hydroxyapatite (HA) chromatography, and CRM197 was eluted with solvents of 100 mM potassium phosphate and 100 mM NaCl. The SDS-PAGE result of eluted CRM197 is shown in FIG. 13(B).

[143]

[144] Example 5: Comparison with CRM197 produced using Corynebacterium

[145] The quality and characteristics of the final purified CRM197 were analyzed. As a result of SEC-HPLC analysis, the purity was found to be 99% or more (FIG. 14). SDS-PAGE and Western blotting showed that the band appeared at the same position as CRM197 obtained using Corynebacterium (FIG. 15).

[146] In addition, the entire 535 amino acid sequence comprising CRM197 was found to be 100% identical, and molecular weight measurements identified a major peak at 58,409 Da, consistent with the theoretical molecular weight (FIG. 16). The higher order structure was identified by circular dichroism (CD) analysis and it was found that there was no difference in the higher order structure with CRM197 produced using Corynebacterium (FIG. 17). Analysis of the fluorescence spectrum showed that the maximum emission wavelength was 338 nm, which was the same as that of CRM197 obtained using Corynebacterium (FIG. 18).

[147] The results described above showed that CRM197 produced using E. coli strain pHex-L3 was physicochemically and immunologically the same as CRM197 produced using Corynebacterium.

[148]

Industrial applicability

[149] The present invention is very suitable for the production of CRM197 protein, since the CRM197 protein, having the same physicochemical/immunological properties as the protein isolated from the parent bacteria, can be expressed in normal E. coli, in which the redox potential is not regulated, and CRM197 protein, which has a high efficiency of secretion into the periplasm, can be obtained without shifting the pH of the environment to increase secretion into the periplasm.

[150]

[151] Although specific configurations of the present invention have been described in detail, those skilled in the art will appreciate that this description is provided to set forth preferred embodiments for illustrative purposes and should not be construed as limiting the scope of the present invention. Thus, the essential scope of the present invention is defined by the appended claims and their equivalents.

[152]

List of sequences in free text form

[153] The electronic file is attached.

--->

LIST OF SEQUENCES

<110> Genofocus, Inc.

<120> PROTEIN EXPRESSION METHOD CRM197

<130> P19-B209

<160> 56

<170> KoPatentIn 3.0

<210> 1

<211> 1226

<212> DNA

<213> Artificial sequence

<220>

<223> TPB1Tv1.3

<400> 1

tactgagcta ataacaggcc tgctggtaat cgcaggcctt tttatttctg gctcaccttc 60

gggtgggcct ttctgcgttt acggttctgg caaatattct gaaatgagct gttgacaatt 120

aatcatccgg ctcgtataat gtgtggaatt gtgagcggat aacaatttac tgctctttaa 180

caatttatca gatccaattg gaggaacaat atgggagacc acgcgcgcga ggtctcatct 240

gcggcggcag cagggggatc gatgagcaaa ggtgaagagc tgtttaccgg tgtggtgccg 300

attctggttg aactggatgg cgacgttaac ggccataaat tcagcgtcag cggcgagggc 360

gaaggggatg ccacctacgg taaactgacc ctgaagttta tttgcaccac cggcaaatta 420

ccggttccgt ggccgacgct ggtgacgacc tttagctatg gcgtgcagtg cttttcccgt 480

tatccggacc atatgaaaca gcatgatttt tttaaaagcg cgatgccgga aggctatgtt 540

caggaacgta ccattttctt taaggatgac ggcaattaca aaacccgcgc ggaagtgaaa 600

tttgaaggtg ataccctggt caaccgcatt gaactgaaag gcattgattt caaagaagat 660

ggtaatattc tcggtcataa gctggaatat aactacaaca gccataacgt ttatatcatg 720

gcggataaac aaaaaaacgg tattaaagtg aactttaaaa ttcgccataa tatcgaagat 780

ggtagcgtgc aactggcgga tcattatcag cagaacaccc caattggcga tggcccggtg 840

ttgctgccgg ataaccacta tctgagcacc cagtcggcgc tgagtaaaga tccgaacgaa 900

aaacgcgatc acatggtgct gctggagttt gtcaccgccg ccggtatcac ccacggcatg 960

gatgaactgt ataaataatg atttaaagcg gatatcaaat aaaacgaaag gctcagtcga 1020

aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa 1080

atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac 1140

gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt 1200

ttgcgtttct acaaactctt tttgtt 1226

<210> 2

<211> 1625

<212> DNA

<213> Artificial sequence

<220>

<223> CRM197ec

<400> 2

gaagacacgg cgctgacgat gttgttgaca gcagcaagag ctttgttatg gagaatttca 60

gcagctatca cggcaccaaa ccgggctatg ttgacagcat tcagaaaggc atccaaaagc 120

cgaaaagcgg tacccagggc aactacgacg atgactggaa agagttttat agcaccgata 180

acaagtacga cgcggcgggc tatagcgttg ataacgaaaa cccgctgagc ggtaaagcgg 240

gtggcgtggt taaggtgacc tacccgggtc tgaccaaagt gctggcgctg aaggttgaca 300

acgcggaaac catcaagaaa gagctgggcc tgagcctgac cgaaccgctg atggagcagg 360

ttggtaccga ggaattcatt aagcgttttg gtgatggtgc gagccgtgtg gttctgagcc 420

tgccgttcgc ggaaggtagc agcagcgtgg agtacatcaa caactgggaa caagcgaaag 480

cgctgagcgt ggagctggaa attaacttcg aaacccgtgg caagcgtggc caggatgcga 540

tgtacgaata tatggcgcaa gcgtgcgcgg gtaaccgtgt gcgtcgtagc gttggtagca 600

gcctgagctg cattaacctg gattgggacg ttatccgtga caagaccaaa accaagatcg 660

aaagcctgaa agagcacggt ccgattaaaa acaagatgag cgagagcccg aacaagaccg 720

tgagcgagga aaaagcgaag cagtacctgg aggaatttca ccaaaccgcg ctggagcacc 780

cggaactgag cgagctgaaa accgtgaccg gcaccaaccc ggttttcgcg ggtgcgaact 840

atgcggcgtg ggcggtgaac gttgcgcagg tgatcgatag cgaaaccgcg gacaacctgg 900

aaaagaccac cgcggcgctg agcatcctgc cgggtattgg cagcgtgatg ggcatcgcgg 960

atggtgcggt tcaccacaac accgaggaaa tcgtggcgca gagcattgcg ctgagcagcc 1020

tgatggttgc gcaagcgatc ccgctggttg gtgagctggt tgacattggt ttcgcggcgt 1080

acaactttgt ggaaagcatc attaacctgt tccaggtggt tcacaacagc tacaaccgtc 1140

cggcgtatag cccggggccac aaaacccaac cgtttctgca cgatggttat gcggtgagct 1200

ggaacaccgt tgaggacagc atcattcgta ccggtttcca gggcgagagc ggtcacgata 1260

tcaagattac cgcggaaaac accccgctgc cgattgcggg cgttctgctg ccgaccattc 1320

cgggtaaact ggacgttaac aaaagcaaga cccacattag cgtgaacggc cgtaagatcc 1380

gtatgcgttg ccgtgcgatt gatggtgacg tgaccttttg ccgtccgaaa agcccggtgt 1440

acgttggtaa cggcgtgcac gcgaacctgc acgttgcgtt ccaccgtagc agcagcgaga 1500

agatccacag caacgaaatt agcagcgaca gcatcggcgt tctgggttat caaaaaaccg 1560

tggatcatac caaagtgaat agcaagctga gcctgttctt cgagattaaa agctctgagg 1620

tcttc 1625

<210> 3

<211> 535

<212> PRT

<213> Artificial sequence

<220>

<223> CRM197

<400> 3

Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn

1 5 10 15

Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln

20 25 30

Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp

35 40 45

Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala Gly

50 55 60

Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly Val

65 70 75 80

Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val

85 90 95

Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu

100 105 110

Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe Gly

115 120 125

Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly Ser

130 135 140

Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser

145 150 155 160

Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp

165 170 175

Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg

180 185 190

Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val

195 200 205

Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly

210 215 220

Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu

225 230 235 240

Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu

245 250 255

His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val

260 265 270

Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val

275 280 285

Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu

290 295 300

Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala

305 310 315 320

Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser

325 330 335

Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp

340 345 350

Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe

355 360 365

Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His

370 375 380

Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn Thr

385 390 395 400

Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His

405 410 415

Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val

420 425 430

Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys Thr

435 440 445

His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala Ile

450 455 460

Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro Val Tyr Val Gly

465 470 475 480

Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser Ser

485 490 495

Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val Leu

500 505 510

Gly Tyr Gln Lys Thr Val Asp His Thr Lys Val Asn Ser Lys Leu Ser

515 520 525

Leu Phe Phe Glu Ile Lys Ser

530 535

<210> 4

<211> 85

<212> DNA

<213> Artificial sequence

<220>

<223>PelB (L1)

<400> 4

ggtctcatat gaaatatctg ttaccgaccg ccgctgccgg actgctgtta ctggcggcgc 60

agccggcgat ggcgggcgag agacc 85

<210> 5

<211> 88

<212> DNA

<213> Artificial sequence

<220>

<223>G8 (L2)

<400> 5

ggtctcatat gaaaaaaagc ctggttctga aagcgtctgt tgcggtggcg acgctggtgc 60

cgatgctgtc gtttgccggc gagagacc 88

<210> 6

<211> 73

<212> DNA

<213> Artificial sequence

<220>

<223> Wp (L3)

<400> 6

ggtctcatat gcgttctgtg attgttgcct tcctgtttgc ctgtagcttt tgcgtgagcg 60

ccggcgagag acc 73

<210> 7

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<223> OmpT (L4)

<400> 7

ggtctcatat gcgtgcgaaa ctgctcggca ttgttctgac caccccgatt gccatttcca 60

gctttgccgg cgagagacc 79

<210> 8

<211> 82

<212> DNA

<213> Artificial sequence

<220>

<223> OmpA (L5)

<400> 8

ggtctcatat gaaaaaaacc gccatcgcca ttgccgttgc cctcgctggc tttgccaccg 60

tggcgcaggc gggcgagaga cc 82

<210> 9

<211> 82

<212> DNA

<213> Artificial sequence

<220>

<223>G4 (L6)

<400> 9

ggtctcatat gaaactgctg aacgtgatca actttgtttt cctgatgttt gtcagcagca 60

gtagttttgc cggcgagaga cc 82

<210> 10

<211> 73

<212> DNA

<213> Artificial sequence

<220>

<223> G3 (L7)

<400> 10

ggtctcatat gaaaaaactg ctgtttgcca ttccgctggt tgtaccgttt tacagccaca 60

gcggcgagag acc 73

<210> 11

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<223> Lpp (L8)

<400> 11

ggtctcatat gaaagcgacg aaactggtgc tgggtgctgt gattctgggc agcacgctgc 60

tggcgggcgg cgagagacc 79

<210> 12

<211> 88

<212> DNA

<213> Artificial sequence

<220>

<223> Gsp (L9)

<400> 12

ggtctcatat gaaaggtctg aataaaatta cctgctgttt actggcggcg ctgctgatgc 60

cgtgcgcggg tcatgcgggc gagagacc 88

<210> 13

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223>PelB (L1)

<400> 13

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> 14

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223>G8 (L2)

<400> 14

Met Lys Lys Ser Leu Val Leu Lys Ala Ser Val Ala Val Ala Thr Leu

1 5 10 15

Val Pro Met Leu Ser Phe Ala

20

<210> 15

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Wp (L3)

<400> 15

Met Arg Ser Val Ile Val Ala Phe Leu Phe Ala Cys Ser Phe Cys Val

1 5 10 15

Ser Ala

<210> 16

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> OmpT (L4)

<400> 16

Met Arg Ala Lys Leu Leu Gly Ile Val Leu Thr Thr Pro Ile Ala Ile

1 5 10 15

Ser Ser Phe Ala

20

<210> 17

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> OmpA (L5)

<400> 17

Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala

1 5 10 15

Thr Val Ala Gln Ala

20

<210> 18

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223>G4 (L6)

<400> 18

Met Lys Leu Leu Asn Val Ile Asn Phe Val Phe Leu Met Phe Val Ser

1 5 10 15

Ser Ser Ser Phe Ala

20

<210> 19

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> G3 (L7)

<400> 19

Met Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser

1 5 10 15

His Ser

<210> 20

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Lpp (L8)

<400> 20

Met Lys Ala Thr Lys Leu Val Leu Gly Ala Val Ile Leu Gly Ser Thr

1 5 10 15

Leu Leu Ala Gly

20

<210> 21

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Gsp (L9)

<400> 21

Met Lys Gly Leu Asn Lys Ile Thr Cys Cys Leu Leu Ala Ala Leu Leu

1 5 10 15

Met Pro Cys Ala Gly His Ala

20

<210> 22

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 22

gcggatccaa gagacaggat gaggatcgtt tcgc 34

<210> 23

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 23

cggatatcaa gcttggaaat gttgaatact catactcttc 40

<210> 24

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 24

gagatccgga gcttatactg agctaataac 30

<210> 25

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 25

gaaaaataaa caaaaacaaa aagagtttg 29

<210> 26

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 26

tacaaactct ttttgttttt gtttattttt c 31

<210> 27

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 27

cctgttatta gctcagtata agctccggat ctcg 34

<210> 28

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 28

aattggagga acaatatgaa atatct 26

<210> 29

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 29

aacatcgtca gcgcccgcca tcgccggct 29

<210> 30

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 30

ttggaggaac aatatgaaaa aaagcct 27

<210> 31

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 31

aacatcgtca gcgccggcaa acgacagcat 30

<210> 32

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 32

ttggaggaac aatatgcgtt ctgtga 26

<210> 33

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 33

aacatcgtca gcgccggcgc tcacgcaa 28

<210> 34

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 34

ttggaggaac aatatgcgtg cgaaact 27

<210> 35

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 35

aacatcgtca gcgccggcaa agctggaaat 30

<210> 36

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 36

ttggaggaac aatatgaaaa aaacc 25

<210> 37

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 37

aacatcgtca gcgcccgcct gcgccacggt 30

<210> 38

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 38

ttggaggaac aatatgaaac tgctga 26

<210> 39

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 39

aacatcgtca gcgccggcaa aactactgct 30

<210> 40

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 40

ttggaggaac aatatgaaaa aactg 25

<210> 41

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 41

acatcgtcag cgccgctgtg gctgtaaaa 29

<210> 42

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 42

ttggaggaac aatatgaaag cgacgaaa 28

<210> 43

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 43

aacatcgtca gcgccgcccg ccagcagcgt 30

<210> 44

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 44

ttggaggaac aatatgaaag gtctgaa 27

<210> 45

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 45

aacatcgtca gcgcccgcat gacccgcgca 30

<210> 46

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 46

agccggcgat ggcgggcgct gacgatg 27

<210> 47

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 47

atgctgtcgt ttgccggcgc tgacgatg 28

<210> 48

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 48

ttgcgtgagc gccggcgctg acgatg 26

<210> 49

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 49

tttccagctt tgccggcgct gacgatg 27

<210> 50

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 50

accgtggcgc aggcgggcgc tgacgatg 28

<210> 51

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 51

agcagtagtt ttgccggcgc tgacgatg 28

<210> 52

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 52

ttttacagcc acagcggcgc tgacgatg 28

<210> 53

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 53

tgctggcggg cggcgctgac gatg 24

<210> 54

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 54

cgcgggtcat gcgggcgctg acgatg 26

<210> 55

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 55

gatatccgct tttcattagc ttttaatctc gaagaa 36

<210> 56

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 56

ggcgcaagcg tgcgcgggta accgtgtgcg 30

<---

Claims (11)

1. A nucleic acid construct for expressing a CRM197 protein, comprising a nucleic acid encoding a signal sequence that contains the sequence of SEQ ID NO: 6 or SEQ ID NO: 8, and a CRM197 protein gene. 2. The nucleic acid design according to claim 1, wherein said nucleic acid is represented by the nucleotide sequence SEQ ID NO: 8. 3. The nucleic acid design according to claim 1, characterized in that the CRM197 protein gene is represented by the nucleotide sequence SEQ ID NO: 2. 4. An expression vector for expressing the CRM197 protein, containing the nucleic acid construct according to claim 1. 5. The expression vector according to claim 4, additionally containing a Trc promoter. 6. A recombinant microorganism for the expression of the CRM197 protein, containing the nucleic acid construct according to claim 1 or the expression vector according to claim 4. 7. A recombinant microorganism according to claim 6, characterized in that said recombinant microorganism is Escherichia coli. 8. A method for producing CRM197 protein, including: (a) cultivating the recombinant microorganism according to claim 6 to obtain the CRM197 protein; And (b) isolating the resulting CRM197 protein. 9. The method according to claim 8, characterized in that step (b) includes isolating the CRM197 protein secreted into the periplasm.

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