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Secretory expression method of carboxypeptidase B

A technology of secreted expression and carboxypeptidase, applied in peptidases, biochemical equipment and methods, enzymes, etc., can solve the problems of low CPB yield, low renaturation efficiency, limited application, etc., achieve easy recovery and purification, and promote solubility. expressive effect

Inactive Publication Date: 2019-01-04
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] At present, there are mainly two types of methods for preparing active CPB: one is directly extracted and activated from the pancreas of pigs or cattle, but the obtained CPB not only has low yield and high cost, but also may be mixed with protease or infectious substances (such as virus ), so the application is limited; the other is to prepare CPB by gene recombination in prokaryotic (mainly E. coli expression system) or eukaryotic
The eukaryotic system has the disadvantages of long fermentation period and high cost, and is not suitable for large-scale industrial production
The prokaryotic expression system is easy to form inclusion bodies, and the active protein must be denatured and refolded. The operation is cumbersome and the refolding efficiency is low; Soluble expression in Escherichia coli, but the intracellular protein needs to break the cell wall, and the process is cumbersome

Method used

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  • Secretory expression method of carboxypeptidase B
  • Secretory expression method of carboxypeptidase B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: Construction of recombinant expression vector

[0043] (1) Signal peptide site-directed mutation

[0044] Plasmid pET20b (with signal peptide PelB) and synthetic signal peptides OmpA, Endoxylanase, OmpT were used as templates, and primers OA-DB-F and OA-DB-R, PB-DB-F and PB-DB- R, End-DB-F and End-DB-R, OT-DB-F and OT-DB-R for PCR;

[0045]PCR reaction system (200 μL): 100 μL of 2×super Pfu PCR Master Mix, 2 μL of each template, 1 μL of upstream and downstream primers, sterilized ddH 2 O 94 μL;

[0046] PCR amplification conditions: pre-denaturation at 95°C for 5min; denaturation at 94°C for 30s, annealing at 59°C for 30s, extension at 72°C for 2min, 30 cycles; extension at 72°C for 10min.

[0047] After the PCR product was recovered with a DNA purification kit, DpnI was added to digest the template, after purification, it was ligated with T4 DNA ligase overnight at 16°C, and 10 μL of the ligated product was transformed into the cloning host JM109 for c...

Embodiment 2

[0100]Example 2: Expression and purification of carboxypeptidase B in Escherichia coli

[0101] 1. Seed culture: Pick a small amount of strains preserved in glycerol tubes, streak them on LB plates (containing 100 μg / mL ampicillin Amp), and culture them overnight in a 37°C incubator. On the next day, single clones were picked, transferred to 15 mL of LB culture medium (containing 100 μg / mL ampicillin Amp), and cultured overnight at 37° C. on a shaker at 200 rpm.

[0102] 2. Shake flask culture: transfer to the secondary bottle with 2% inoculum, and continue to culture at 37°C until OD 600 When the concentration is 0.5-0.6, add IPTG with a final concentration of 0.1mM, and culture at 25°C for 30h.

[0103] 3. Enzyme hydrolysis of procarboxypeptidase B: centrifuge the induced expression medium at 12,000 rpm for 10 min, and the supernatant is the crude enzyme solution of procarboxypeptidase B, and measure the protein concentration by Coomassie brilliant blue method. Filter the ...

Embodiment 3

[0108] Embodiment 3: the enzyme activity assay of recombinant CPB

[0109] Assay buffer: 25mM Tris-HCl, containing 0.1mol / L NaCl (pH7.65)

[0110] Substrate solution (1.0mmol / L): Accurately weigh 16.7mg of HIPPURYL-L-ARGININE (Sigma), add 50mL of bioassay buffer to dissolve it, or dissolve it in equal proportions according to the actual weight weighed. After the configuration is completed, it is placed for 30 minutes to ensure that it is fully dissolved, and it is a 1.0mmol / L substrate solution. Ready to use. Preheat to 25°C+0.5°C.

[0111] Activity measurement: absorb 3.0mL of substrate solution, take a covered quartz cuvette with an optical path of 1cm, add 3.0mL of substrate solution preheated at 25°C, and immediately adjust to zero at a wavelength of 254nm. Precisely draw 10 μl of the recombinant CPB enzyme solution and add it, time it immediately and shake it well, and read the absorbance every 30 seconds for a total of 5 minutes. Measure three times and take the aver...

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Abstract

The invention discloses a secretory expression method of carboxypeptidase B. The secretory expression method of the carboxypeptidase B includes: (1) adopting sequences of signal peptides including OmpA, PelB, Endoxylanase and OmpT as starting sequences to perform site-specific mutagenesis; (2) preparing a nucleotide sequence SDB-pCPB-6xHis; (3) preparing recombinant expression vectors; (4) transforming the recombinant expression vectors into host escherichia coli E.coli BL21 and induce to express pCPB; (5) enzyme-digesting and purifying the pCPB. The secretory expression method of the carboxypeptidase B has the advantages that through transformation of the signal peptides, the procarboxypeptidase B can be directly secreted into a culture medium, compared with intracellular and periplasmicexpressions, the secretory expression method does not need smudge cells, and recovery and purification of the carboxypeptidase B are easier to operate. In addition, the culture medium with enough space is beneficial to overexpression of recombinant protein.

Description

technical field [0001] The invention relates to the technical field of gene expression, in particular to a method for adding a signal peptide mutant to the N-terminus of the procarboxypeptidase B gene to make the target protein soluble secreted and expressed in Escherichia coli. Background technique [0002] Carboxypeptidase B (CPB for short, EC 3.4.17.2) is a metalloenzyme containing zinc ions, which can specifically remove basic amino acids at the C-terminus of proteins or polypeptides, especially arginine and lysine . Carboxypeptidase B contains 307 amino acids with a molecular weight of 35 kDa. Procarboxypeptidase B is the zymogen form of carboxypeptidase B, and trypsin can activate procarboxypeptidase B to obtain active carboxypeptidase B. Carboxypeptidase B has a wide range of applications, mainly for scientific research, medical diagnosis and drug production. In particular, carboxypeptidase B is one of the indispensable dual enzymes in the process of proinsulin act...

Claims

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Application Information

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IPC IPC(8): C12N9/48C12N15/62C12N15/70
CPCC07K2319/02C07K2319/21C12N9/485C12N15/70C12Y304/17002
Inventor 张梁於瑞梅辛瑜顾正华李由然丁重阳石贵阳
Owner JIANGNAN UNIV
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