A genetically engineered bacterium with high expression of clostridial protease and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a genetically engineered bacterium with high expression of clostridial protease and application thereof. The genetically engineered bacterium provided by the application co-expresses clostridial protease and a molecular chaperone; the molecular chaperone is GroEL and KAR2; the nucleotide sequence of the clostridial protease is shown as SEQ ID NO. 2; and the molecular chaperone has the nucleotide sequences shown as SEQ ID NO. 6 and SEQ ID NO. 10. The genetically engineered bacterium with co-expression of clostridial protease and a molecular chaperone provided by the application enables efficient expression of Arg-C in a soluble form, thereby efficiently and stably producing Arg-C. The obtained Arg-C can decompose complex proteins into specific peptide segments, so that the presence and content of a marker can be more accurately determined.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a genetically engineered bacterium with high expression of clostripain and application thereof. BACKGROUND

[0002] Clostripain, also known as Arg-C, is a protease extracted from Clostridium histolyticum, which belongs to endo-type enzymes and can specifically recognize and cut the specific amino acid sequence (such as the carboxyl terminal peptide bond of arginine residue) in the peptide chain. It plays an important role in protein structure research, recombinant protein purification, etc. and is commonly used in scientific research experiments and also applied to the treatment of some diseases, such as activating or inactivating some proteins with physiological activity by using its cutting characteristics. Clostripain can be used to process samples and decompose complex tumor-related proteins into specific peptide segments. By detecting these peptide segments, the presence and content of tumor markers can be more accurately determined. For example, in the detection of alpha-fetoprotein, a liver cancer-related protein, the specific cutting of clostripain can expose the hidden antigen epitope, thereby improving the sensitivity and accuracy of the detection. In the detection of myocardial injury markers (such as troponin and myoglobin), impurity proteins in the sample may interfere with the detection. Clostripain can remove these impurity proteins and may also cut the marker proteins appropriately to facilitate their recognition by detection reagents, thereby assisting in the early diagnosis of cardiovascular diseases. It can be seen that clostripain has good application prospects in the field of in vitro diagnostic technology.

[0003] However, the defects of clostripain limit its application. On the one hand, the stability of enzyme activity is limited. The activity of clostripain is sensitive to environmental conditions such as temperature and pH. When the temperature is slightly increased or the pH deviates from the optimum range, the activity of the enzyme may decrease significantly or even be inactivated, which limits the application of clostripain in some reaction systems with high requirements. On the other hand, the cost of clostripain is relatively high. The production and purification process of clostripain is complex, which makes the cost factor one of the reasons that limits its widespread use in large-scale applications.

[0004] The production of functional enzymes and proteins, whole-cell catalysts for the biosynthesis of compounds, high-throughput synthesis and testing of artificially designed proteins, etc. have important applications in the fields of biocatalysis and biosynthesis. The above fields all rely on the efficient expression of heterologous proteins in a soluble form with a natural structure in prokaryotic hosts. However, due to the protein synthesis and folding system of prokaryotic hosts, many proteins form inactive inclusion bodies when expressed, which limits the industrial application of prokaryotic host protein expression systems. The reason is that the exogenously expressed proteins fail to fold correctly in prokaryotic hosts, thereby affecting their structure and function. Therefore, new solutions are urgently needed to solve this problem. SUMMARY

[0005] In view of the above, the present application provides

[0006] The genetically engineered bacteria provided by the present application co-express clostridial protease and chaperone; the chaperone is GroEL and KAR2; the nucleotide sequence of the clostridial protease is shown in SEQ ID NO. 2; the chaperone has the nucleotide sequence shown in SEQ ID NO. 6 and SEQ ID NO. 10. The genetically engineered bacteria co-expressing clostridial protease and chaperone provided by the present application can efficiently express Arg-C in a soluble form, thereby efficiently and stably producing Arg-C. The obtained Arg-C can decompose complex proteins into specific peptide segments, thereby more accurately judging the presence and content of markers.

[0007] The technical scheme of the present application comprises:

[0008] In one aspect, the present application provides a genetically engineered bacteria with high expression of Arg-C, which co-expresses clostridial protease and chaperone; the chaperone is GroEL and KAR2.

[0009] Preferably, the nucleotide sequence of the clostridial protease is shown in SEQ ID NO. 2;

[0010] SEQ ID NO. 2:

[0011]

[0012] In particular, the chaperone has a nucleotide sequence as shown in SEQ ID NO. 6 and SEQ ID NO. 10.

[0013] Preferably, the chaperone GroEL has a nucleotide sequence as shown in SEQ ID NO. 6.

[0014] SEQ ID NO. 6:

[0015]

[0016] Preferably, said chaperone KAR2 has a nucleotide sequence as set forth in SEQ ID NO. 10.

[0017] SEQ ID NO. 10:

[0018]

[0019] Specifically, the preparation method of the genetically engineered bacteria comprises the following steps:

[0020] S1, amplifying a gene fragment as shown in SEQ ID NO. 2, SEQ ID NO. 6 or SEQ ID NO. 10 to obtain an Arg-C fragment, a GroEL fragment or a KAR2 fragment;

[0021] S2, respectively cloning the Arg-C fragment, the GroEL fragment or the KAR2 fragment with the enzyme-recovered carrier fragment;

[0022] S3, respectively transforming a competent cell, screening a positive clone, and obtaining an Arg-C expression plasmid, a GroEL expression plasmid or a KAR2 expression plasmid;

[0023] S4, transforming a host cell after enzyme cutting of the Arg-C expression plasmid, screening a positive clone, and obtaining an engineered bacterium expressing clostridial protease;

[0024] S5, mixing the enzyme-cut GroEL expression plasmid with the enzyme-cut KAR2 expression plasmid to obtain an enzyme-cut mixed solution;

[0025] S6, transforming the engineered bacterium expressing clostridial protease with the enzyme-cut mixed solution, screening a positive clone, and obtaining a genetically engineered bacterium.

[0026] Preferably, the nucleotide sequence of the primer used for amplification in step S1 is as shown in SEQ ID NO. 3-4, SEQ ID NO. 7-8 and SEQ ID NO. 11-12.

[0027] Further preferably, the nucleotide sequence of the primer used for amplification of SEQ ID NO. 2 in step S1 is as shown in SEQ ID NO. 3-4;

[0028] SEQ ID NO. 3: ATGCTGCGAAGAAAGGTTAGTACT.

[0029] SEQ ID NO. 4: CCACTGGTAGTGGTTAACAC.

[0030] Further preferably, the nucleotide sequence of the primer used for amplification of SEQ ID NO. 6 in step S1 is as shown in SEQ ID NO. 7-8;

[0031] SEQ ID NO. 7: ATGGCAAAAGAGATTAAGTTTAGTG.

[0032] SEQ ID NO. 8: CATCATTCCTCCCATATCAGG.

[0033] Further preferably, the nucleotide sequence of the primers used for amplifying SEQ ID NO. 10 in step S1 is shown as SEQ ID NO. 11-12.

[0034] SEQ ID NO. 11: ATGTTTTCCGCAAGAAAATCTTCCGTAGG.

[0035] SEQ ID NO. 12: TCTAGCTTCACTTTTAGCCTTGAACTTGG.

[0036] Preferably, the vector in step S2 comprises one or more of pPIC9, pHIL-S1, pPICZ alpha, pYAM75P, pPIC9K, pPIC3.5K, pGAPZ, pPIC3, pPICZ, pPSC3K, pHIL-D2.

[0037] Further preferably, the vector in step S2 comprises one or more of pPIC9, pGAPZ, pHIL-S1.

[0038] Still further preferably, the Arg-C fragment is cloned and constructed with the pPIC9 vector fragment recovered by enzyme digestion.

[0039] Still further preferably, the GroEL fragment is cloned and constructed with the pGAPZ vector fragment recovered by enzyme digestion.

[0040] Still further preferably, the KAR2 fragment is cloned and constructed with the pHIL-S1 vector fragment recovered by enzyme digestion.

[0041] Preferably, the competent cell in step S3 is selected from one or more of DH-5a competent cell, TG1 competent cell, JM109 competent cell.

[0042] Further preferably, the competent cell in step S3 is DH-5a competent cell.

[0043] Preferably, the host cell in step S4 is Pichia pastoris GS115 competent cell.

[0044] Specifically, the concentration ratio of the GroEL expression plasmid after enzyme digestion to the KAR2 expression plasmid after enzyme digestion in step S5 is 1:1-2.

[0045] Preferably, the concentration ratio of the enzyme-digested GroEL expression plasmid to the enzyme-digested KAR2 expression plasmid in step S5 is 1:1.5.

[0046] In another aspect, the present application provides a method for preparing the genetically engineered bacteria as described in any of the above.

[0047] In yet another aspect, the present application provides a method for producing Arg-C, which comprises using the genetically engineered bacteria or the Arg-C produced by the method as described in any of the above.

[0048] In yet another aspect, the present application provides an immunoassay reagent, which comprises the genetically engineered bacteria or the Arg-C produced by the method as described in any of the above.

[0049] In yet another aspect, the genetically engineered bacteria, the Arg-C produced by the method or the immunoassay reagent as described in any of the above are used in the preparation of an immunoassay product.

[0050] Preferably, the immunoassay product comprises an immunoassay kit or an immunoassay chip.

[0051] In yet another aspect, the present application provides an immunoassay kit, which comprises the genetically engineered bacteria, the Arg-C produced by the method or the immunoassay reagent as described in any of the above.

[0052] In yet another aspect, the present application provides an immunoassay chip, which comprises the genetically engineered bacteria, the Arg-C produced by the method or the immunoassay reagent as described in any of the above.

[0053] In yet another aspect, the present application provides an immunoassay method, which comprises using the genetically engineered bacteria, the Arg-C produced by the method, the immunoassay reagent, the immunoassay kit or the immunoassay chip as described in any of the above.

[0054] Specifically, the immunoassay method comprises pre-treating a sample, which comprises the following steps:

[0055] (1) mixing the genetically engineered bacteria, the Arg-C produced by the method or the immunoassay reagent with a sample to be tested, and performing an enzymatic reaction;

[0056] (2) adding an enzyme reaction termination solution, and separating and collecting a target product;

[0057] (3) performing an immunoassay on the target product.

[0058] Preferably, the sample to be tested in step (1) comprises one or more of serum, plasma, urine, cell culture supernatant and tissue samples.

[0059] Preferably, the immunoassay in step (3) comprises one or more of immunoblotting detection, enzyme-linked immunosorbent assay, chemiluminescence immunoassay.

[0060] The beneficial effects of the present application are:

[0061] The genetically engineered bacteria provided by the present application co-expresses clostridial protease and chaperone; the chaperone is GroEL and KAR2; the nucleotide sequence of the clostridial protease is shown in SEQ ID NO. 2; the chaperone has the nucleotide sequence shown in SEQ ID NO. 6 and SEQ ID NO. 10. The genetically engineered bacteria co-expressing clostridial protease and chaperone of the present application enables efficient expression of Arg-C in soluble form, thereby efficiently and stably producing Arg-C. The obtained Arg-C can decompose complex proteins into specific peptide segments, thereby more accurately determining the presence and content of markers. DETAILED DESCRIPTION

[0062] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are used. If the reagents or instruments used are not specified by the manufacturers, they are all conventional products that can be purchased in the market. The features and performances of the present application will be described in further detail below in combination with the embodiments.

[0063] Example 1 Construction of pPIC9-Arg-C recombinant plasmid

[0064] 1. Construction of Arg-C fragment

[0065] Clostridial protease Arg-C derived from Clostridium histolyticum has an amino acid sequence as shown in SEQ ID NO. 1. The codons of the amino acid sequence shown in SEQ ID NO. 1 are optimized to obtain the nucleotide sequence shown in SEQ ID NO. 2. The sequence optimized by codons is genetically synthesized by a gene synthesis unit. The Arg-C fragment is obtained by PCR amplification using primer pairs (Arg-C-F, Arg-C-R).

[0066] SEQ ID NO. 1:

[0067] MLRRKVSTLLMTALITTSFLNSKPVYANPVTKSKDNNLKEVQQVTSKSNKNKNQKVTIM YYCDADNNLEGSLLNDIEEMKTGYKDSPNLNLIALVDRSPRYSSDEKVLGEDFSDTRLY KIEHNKANRLDGKNEFPEISTTSKYEANMGDPEVLKKFIDYCKSNYEADKYVLIMANHG GGAREKSNPRLNRAICWDDSNLDKNGEADCLYMGEISDHLTEKQSVDLLAFDACLMGT AEVAYQYRPGNGGFSADTLVASSPVVWGPGFKYDKIFDRIKAGGGTNNEDDLTLGGKE QNFDPATITNEQLGALFVEEQRDSTHANGRYDQHLSFYDLKKAESVKRAIDNLAVNLSN ENKKSEIEKLRGSGIHTDLMHYFDEYSEGEWVEYPYFDVYDLCEKINKSENFSSKTKD LASNAMNKLNEMIVYSFGDPSNNFKEGKNGLSIFLPNGDKKYSTYYTSTKIPHWTMQ SWYNSIDTVKYGLNPYGKLSWCKDGQDPEINKVGNWFELLDSWFDKTNDVTGGVNHY QW.

[0068] The sequence information of the primer pair (Arg-C-F, Arg-C-R) is shown in Table 1:

[0069] Table 1 Sequence information of primer pair

[0070]

[0071] Note: "F" in the table represents forward primer, and "R" represents reverse primer.

[0072] 2. Construction of pPIC9-Arg-C recombinant plasmid

[0073] 1) After extracting the pPIC9 plasmid, 50 μL of the plasmid was added to 30 μL of water, 10 μL of enzyme (NcoI: XhoI = 1:1 v / v) and 10 μL of 10X buffer were added, and after mixing, incubation at 37°C for 3 h, vector recovery was performed to obtain the enzyme-digested recovered pPIC9 vector fragment.

[0074] 2) The pPIC9-Arg-C recombinant plasmid was constructed using the GBclonart Seamless Cloning Kit (Suzhou Shen Zhou Gene Co., Ltd.). The specific steps are as follows:

[0075] (1) The cloning reaction solution was prepared according to the reaction system in Table 2:

[0076] Table 2 Reaction system

[0077]

[0078] After the cloning reaction solution was mixed evenly, it was placed in a 45°C water bath for 30 min and then transferred to ice. 5 μL of DH-5α competent cells were added to the cloning reaction solution, which was then placed on ice for 5 min, in a 42°C water bath for 1 min, and on ice for 2 min. 400 μL of LB medium was added, and the solution was incubated at 37°C for 60 min, centrifuged, and evenly spread on an LB plate containing 100 mg / L Zeocin. The next day, the clones were picked and incubated overnight, and the plasmid was extracted to obtain the pPIC9-Arg-C recombinant plasmid. After the construction of the pPIC9-Arg-C recombinant plasmid was completed, the whole gene was sequenced by a gene synthesis unit to verify the integrity of the recombinant plasmid.

[0079] Example 2 Construction of pGAPZA-GroEL recombinant plasmid

[0080] 1. Construction of GroEL fragment

[0081] GroEL has an amino acid sequence as set forth in SEQ ID NO. 9. The codons of the amino acid sequence set forth in SEQ ID NO. 5 were optimized to obtain the nucleotide fragment set forth in SEQ ID NO. 6. The codon-optimized sequence was synthesized by a gene synthesis unit, and PCR amplification was performed using a primer pair (GroEL-F, GroEL-R) to obtain the GroEL fragment.

[0082] SEQ ID NO. 5:

[0083] MAKEIKFSEEARRAMLRGVDKLADAVKVTLGPKGRNVVLEKKFGSPLITNDGVTIAKEIELEDPFENMGAKLVAEVASKTNDVAGDGTTTATVLAQAMIREGLKNVTAGANPMGIRKGIEKAVAVAVEELKAISKPIKGKESIAQVAAISAADEEVGQLIAEAMERVGNDGVITLEESKGFTTELDVVEGMQFDRGYVSPYMITDTEKMEAVLENPYILITDKKISNIQDILPILEQVVQQGKPLLIIAEDVEGEALATLVVNKLRGTFTAVAVKAPGFGDRRKAMLEDIAILTGGEVISEELGRDLKSTTIASLGRASKVVVTKENTTIVDGAGDSERIKARINQIRAQLEETTSEFDREKLQERLAKLAGGVAVIKVGAATETELKERKLRIEDALNSTRAAVEEGIVAGGGTALMNVYNKVAAIEAEGDEATGVKIVLRAIEEPVRQIAQNAGLEGSVIVERLKTEKPGIGFNAATGEWVDMIEAGIVDPTKVTRSALQNAASVAAMFLTTEAVVADKPEENKGGNPGMPDMGGMM.

[0084] The sequence information of the primer pair (GroEL-F, GroEL-R) is shown in Table 2:

[0085] Table 2 Sequence information of primer pair

[0086]

[0087] Note: "F" in the table represents forward primer, and "R" represents reverse primer.

[0088] 2. Construction of pGAPZA recombinant plasmid

[0089] According to the method of "2. Construction of pPIC9-Arg-C recombinant plasmid" in Example 1, the pGAPZA-GroEL recombinant plasmid was constructed. After the construction of the pGAPZA-GroEL recombinant plasmid was completed, the whole gene sequencing was performed by the gene synthesis unit to verify the integrity of the recombinant plasmid.

[0090] Example 3. Construction of pHIL-S1-KAR2 recombinant plasmid

[0091] 1. Construction of KAR2 fragment

[0092] KAR2 has an amino acid sequence as shown in SEQ ID NO. 9. The amino acid sequence shown in SEQ ID NO. 9 is codon-optimized to obtain a nucleotide fragment shown in SEQ ID NO. 10. The codon-optimized sequence is genetically synthesized by a genetic synthesis unit, and a primer pair (KAR2-F, KAR2-R) is used for PCR amplification to obtain a KAR2 fragment.

[0093] SEQ ID NO. 9:

[0094] MFSARKSSVGWLVSSLAVFYVLLAVIMPIALTGSQSSRVVARAAEDHEDYGTVIGIDLGTTYSCVAVMKNGKTEILANEQGNRITPSYVSFTDDERLIGDAAKNQAASNPKNTIFDIKRLIGLQYNDPTVQRDIKHLPYTVVNKGNKPYVEVTVKGEKKEFTPEEVSGMILGKMKQIAEDYLGKKVTHAVVTVPAYFNDAQRQATKDAGAIAGLNILRIVNEPTAAAIAYGLDKTEDEHQIIVYDLGGGTFDVSLLSIENGVFEVQATAGDTHLGGEDFDYKLVRHFAQLFQKKHDLDVTKNDKAMAKLKREAEKAKRSLSSQTSTRIEIDSFFNGIDFSETLTRAKFEELNLALFKKTLKPVEKVLKDSGLQKEDIDDIVLVGGSTRIPKVQQLLEKFFNGKKASKGINPDEAVAYGAAVQAGVLSGEEGVEDIVLLDVNALTLGIETTGGVMTPLIKRNTAIPTKKSQIFSTAVDNQKAVRIQVYEGERAMVKDNNLLGNFELSDIRAAPRGVPQIEVTFALDANGILTVSATDKDTGKSESITIANDKGRLSQDDIDRMVEEAEKYAAEDAKFKAKSEAR.

[0095] The sequence information of the primer pair (KAR2-F, KAR2-R) is shown in Table 3:

[0096] Table 3 Sequence information of primer pair

[0097]

[0098] Note: In the table, “F” represents a forward primer, and “R” represents a reverse primer.

[0099] 2. Construction of pHIL-S1-KAR2 recombinant plasmid

[0100] The pHIL-S1-KAR2 recombinant plasmid was constructed according to the method of "2. Construction of pPIC9-Arg-C recombinant plasmid" in Example 1. After the construction of the pHIL-S1-KAR2 recombinant plasmid, the whole gene sequencing was performed by the gene synthesis unit to verify the integrity of the recombinant plasmid.

[0101] Example 4. Genetically engineered bacteria P. pastaris-pPIC9-Arg-C

[0102] 1. 10 μg of the recombinant plasmid pPIC9-Arg-C prepared in Example 1 was linearized by Sac I enzyme, and was recovered by precipitation with 2 volumes of anhydrous ethanol, and was dissolved in 30 μL of ddH2O. 100 μL of competent cells of P. pastoris (P. pastaris) GS115 was added to 30 μL of the linearized enzyme-digested plasmid, and after mixing, it was transferred into an electroporation cup, and was placed on ice for several minutes, and then was subjected to electroporation (voltage 1680 V, pulse time 5 mS) using an electroporator. Immediately after the electroporation, 1 ml of D-sorbitol (IM) was added, and it was placed on ice for several minutes, and then the bacterial solution was spread on a YPD plate containing 100 μg / mL of G418, and was cultured in a 28°C incubator. After the antibiotic selection and PCR verification, the genetically engineered bacteria P. pastaris-pPIC9-Arg-C was obtained.

[0103] Example 5. Genetically engineered bacteria P. pastaris-Arg-C-GroEL-KAR2

[0104] 1. 2 μg of the recombinant plasmid pGAPZA-GroEL prepared in Example 2 was linearized by Sac I enzyme, and was recovered by precipitation with 2 volumes of anhydrous ethanol, and was dissolved in 15 μL of ddH2O. 3 μg of the recombinant plasmid pHIL-S1-KAR2 prepared in Example 3 was linearized by Sac I enzyme, and was recovered by precipitation with 2 volumes of anhydrous ethanol, and was dissolved in 15 μL of ddH2O. After mixing, the enzyme-digested mixed solution was obtained.

[0105] 2, Take 100 μL of genetically engineered bacteria P. pastaris-pPIC9-Arg-C competent cells, add 30 μL of enzyme cutting mixed solution, mix and transfer into the electric conversion cup, place on ice for a few minutes, then put into the electric conversion instrument for electric shock (voltage 1680V, electric shock time 5mS), immediately add 1 mL of D-sorbitol (IM) after electric shock, place on ice for a few minutes, then spread the bacterial solution on the YPD plate containing 100 μg / mL Zeocin, and place in the 28°C incubator for culture. After antibiotic screening and PCR verification, the genetically engineered bacteria P. pastaris-Arg-C-GroEL-KAR2 are obtained.

[0106] Comparative Example 1 Genetically engineered bacteria P. pastaris-Arg-C-GroEL

[0107] 1, 5 μg of the recombinant plasmid pGAPZA-GroEL prepared in Example 2 is linearized and cut with Sac I enzyme, precipitated and recovered with 2 volumes of anhydrous ethanol, dissolved in 30 μL of ddH2O to obtain the enzyme cutting solution.

[0108] 2, Take 100 μL of genetically engineered bacteria P. pastaris-pPIC9-Arg-C competent cells, add 30 μL of enzyme cutting mixed solution, mix and transfer into the electric conversion cup, place on ice for a few minutes, then put into the electric conversion instrument for electric shock (voltage 1680V, electric shock time 5mS), immediately add 1 mL of D-sorbitol (IM) after electric shock, place on ice for a few minutes, then spread the bacterial solution on the YPD plate containing 100 μg / mL Zeocin, and place in the 28°C incubator for culture. After antibiotic screening and PCR verification, the genetically engineered bacteria P. pastaris-Arg-C-GroEL-KAR2 are obtained.

[0109] Comparative Example 2 Genetically engineered bacteria P. pastaris-Arg-C-KAR2

[0110] 1, 5 μg of the recombinant plasmid pHIL-S1-KAR2 prepared in Example 3 is linearized and cut with Sac I enzyme, precipitated and recovered with 2 volumes of anhydrous ethanol, dissolved in 30 μL of ddH2O to obtain the enzyme cutting solution.

[0111] 2. Take 100 μL of the genetically engineered bacteria P. pastaris-pPIC9-Arg-C competent cells, add 30 μL of enzyme digestion solution, mix, and then transfer into an electroporation cup. Place on ice for several minutes, then place into an electroporation instrument for electroporation (voltage 1680 V, pulse time 5 mS). Immediately after electroporation, add 1 mL of D-sorbitol (IM), place on ice for several minutes, then spread the bacterial solution onto a YPD plate containing 100 μg / mL Zeocin, and place in a 28°C incubator for culture. After antibiotic screening and PCR verification, obtain the genetically engineered bacteria P. pastaris-Arg-C-KAR2.

[0112] Example 1 Concentration and activity of clostridial protease in genetically engineered bacteria

[0113] After the genetically engineered bacteria strains P. pastaris-pPIC9-Arg-C of Example 4, P. pastaris-Arg-C-GroEL-KAR2 of Example 5, P. pastaris-Arg-C-GroEL of Comparative Example 1, or P. pastaris-Arg-C-KAR2 of Comparative Example 2 were inoculated onto YPD plates and cultured at 30°C for 48 h, single colonies were picked and inoculated into 250 mL shake flasks containing 50 mL of YPD medium, and cultured at 30°C and 220 r / min until the OD600=7. The fermentation broth was centrifuged at 5000 r / min for 5 min, the supernatant was removed, and the bacterial pellet was washed twice with sterile physiological saline. The bacterial pellet was resuspended with 50 mL of BMMY medium at pH 6.0, and the fermentation was continued at 30°C and 220 r / min, with 1% methanol added every 24 h. After 120 h of fermentation, the fermentation supernatant was taken, and the concentration and activity of clostridial protease were determined, as follows:

[0114] 1. Concentration determination:

[0115] The fermentation supernatant of P. pastaris-pPIC9-Arg-C, P. pastaris-Arg-C-GroEL-KAR2, P. pastaris-Arg-C-GroEL, or P. pastaris-Arg-C-KAR2 was appropriately diluted 50-fold with sterile physiological saline, 5 mL of Bradford reagent was added, mixed, and then allowed to stand at room temperature for 5 min. The OD value was determined at 595 nm, and the protein concentration was calculated according to the standard curve.

[0116] 2. Activity determination:

[0117] The casein was prepared into 5 mg / mL substrate solution with Tris-HCl buffer solution of pH 7.5, and preheated at 37°C. 1 mL of preheated substrate was added with 100 μL of P. pastaris-pPIC9-Arg-C, P. pastaris-Arg-C-GroEL-KAR2, P. pastaris-Arg-C-GroEL or P. pastaris-Arg-C-KAR2 fermentation supernatant, respectively, and reacted at 37°C for 15 min, then 2 mL of 5% trichloroacetic acid was added, mixed, and left at room temperature for 10 min, centrifuged at 10,000 rpm for 10 min, and the supernatant was taken. 1 mL of supernatant was added with 5 mL of 0.4 mol / L sodium carbonate solution, and then 0.5 mL of Folin-phenol reagent (diluted 1:10 before use) was added, mixed, and incubated at 37°C for 20 min, and the absorbance (OD value) was measured at 660 nm. The enzyme activity was calculated according to the standard curve.

[0118] The results are shown in Table 4.

[0119] Table 4 Clostridial protease concentration and activity

[0120]

[0121] The results show that P. pastaris-Arg-C-GroEL-KAR2 can significantly improve the concentration and activity of clostridial protease, indicating that the molecular chaperone GroEL and KAR2 can promote the secretion and expression of clostridial protease, and have a synergistic effect.

[0122] The above detailed description is a specific description of one of the feasible embodiments of the present application, which is not used to limit the patent scope of the present application. It should be noted that any equivalent implementation or change made without departing from the present application should be included in the scope of the technical solutions of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A genetically engineered bacteria with high expression of Arg-C, characterized in that, The genetically engineered bacteria co-express clostridial protease and molecular chaperone; the molecular chaperone is GroEL and KAR2; the amino acid sequence of the clostridial protease is shown in SEQ ID NO. 1; the amino acid sequence of the GroEL is shown in SEQ ID NO. 5; the amino acid sequence of the KAR2 is shown in SEQ ID NO. 9; and the host cell of the genetically engineered bacteria is Pichia pastoris GS115 competent cell. 2.The genetically engineered bacteria according to claim 1, characterized in that, The nucleotide sequence of the clostridial protease is shown in SEQ ID NO. 2; and the nucleotide sequence of the molecular chaperone is shown in SEQ ID NO. 6 and SEQ ID NO.

10.

3. The method for preparing the genetically engineered bacteria according to any one of claims 1 to 2, characterized in that, The preparation method comprises the following steps: S1, amplifying the gene fragment shown in SEQ ID NO. 2, SEQ ID NO. 6 or SEQ ID NO. 10 to obtain Arg-C fragment, GroEL fragment or KAR2 fragment; S2, respectively cloning the Arg-C fragment, GroEL fragment or KAR2 fragment and the vector fragment recovered by enzyme digestion; S3, respectively transforming the competent cells and screening the positive clones to obtain Arg-C expression plasmid, GroEL expression plasmid or KAR2 expression plasmid; S4, transforming the host cell after enzyme digestion of the Arg-C expression plasmid and screening the positive clones to obtain the engineered bacteria expressing clostridial protease; S5, mixing the GroEL expression plasmid after enzyme digestion and the KAR2 expression plasmid after enzyme digestion to obtain an enzyme digestion mixed solution; S6, transforming the engineered bacteria expressing clostridial protease with the enzyme digestion mixed solution and screening the positive clones to obtain the genetically engineered bacteria.

4. The genetically engineered bacteria according to any one of claims 1-2 for use in producing Arg-C.

5. A method for producing Arg-C, characterized by, The production method comprises using the genetically engineered bacteria according to any one of claims 1-2.

Images