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Method of producing mutation procarboxypeptidase B and mutation carboxypeptidase B

A technology of pro-carboxypeptidase and carboxypeptidase, which is applied in the field of molecular biology and can solve the problem that the mutation of CPB by site-directed mutagenesis technology is not reported.

Inactive Publication Date: 2007-10-24
BEIJING GUANHONG TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] At present, there is no report in this field on the research of mutating CPB by site-directed mutagenesis technology

Method used

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  • Method of producing mutation procarboxypeptidase B and mutation carboxypeptidase B
  • Method of producing mutation procarboxypeptidase B and mutation carboxypeptidase B
  • Method of producing mutation procarboxypeptidase B and mutation carboxypeptidase B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Construction of mCPB / mPCPB expression plasmid pET-mCPB / pET-mPCPB

[0077] I. The mPCPB gene is derived from the constructed plasmid pET-21a-pCPB containing the procarboxypeptidase B gene, which is obtained from the pancreas of Wistar rats by RT-PCR and obtained after site-directed mutation. (As shown in Figure 1).

[0078] PCR amplification mCPB / mPCPB gene, the DNA nucleotide sequence of three primers used in the cloning: be respectively the 5' end primer of PCPB and the 3' end primer (primer 1 (SEQ ID NO: 3) and primer 3 (SEQ ID NO: 3) IDNO: 5)); CPB's 5' end primer and 3' end primer (primer 2 (SEQ ID NO: 4) and primer 3 (SEQ ID NO: 5)), and another antisense primer was used to introduce Hind III restriction site Stop codon after dot (SEQ ID NO: 6). Nco I / Hind III double restriction site, Nco I / Hind III double restriction site and stop codon were introduced respectively.

[0079] Primer 1: pCPB: sense primer, 5'-GCG GGA TCC CAT GCT TCC GAGGAG CAC TTT GAT ...

Embodiment 2

[0095] Example 2: Screening of recombinant mutant mPCPB clones by inducing expression of mutants by SDS-PAGE

[0096] I. Medium

[0097] After transformation, Escherichia coli BL21(DE3) carrying the recombinant plasmid pET-mPCPB can grow on solid LB medium containing ampicillin (100 μg / ml).

[0098] II. Inoculum

[0099] Pick a single colony and put it in 5ml LB (containing 100μg / ml ampicillin) liquid medium, shake the bacteria overnight at 37°C; inoculate overnight bacteria at 1% in 250ml LB ampicillin-resistant culture medium, shake the bacteria at 37°C The OD at 600nm is 0.3-0.5, induced by 0.5mM IPTG, and adjusted to the corresponding temperature. After 3-5 hours after induction, the bacteria are collected by centrifugation.

[0100] III. Expression Analysis

[0101] Centrifuge the cells before and after induction, discard the supernatant, add electrophoresis sample buffer to the precipitated cells, boil for 10 min, centrifuge, and analyze the supernatant by denaturing ...

Embodiment 3

[0102] Example 3: Induced expression of mutants SDS-PAGE for screening of recombinant mutant mCPB clones

[0103] The screening method is the same as in Example 2. The results are shown in Figure 7.

[0104] Compared with before induction, the corresponding protein band (35KD) was expressed after being induced by 0.5mM IPTG containing the recombinant plasmid. The results showed that strains 1 and 3 were positive clones, and strain 2 was a negative clone.

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Abstract

The invention discloses a saltatory carboxypeptidase B or corresponding procarboxypeptidase B as well as its coded recombinant generating method of polynucleotide, expressive carrier, host cell and carboxypeptidase B or procarboxypeptidase B, wherein the 288th cysteine at natural procarboxypeptidase B is substituted by natural amino acids expect the cysteine, which keeps the activity of natural carboxypeptidase B; the invention also provides an instrument enzyme to prepare saltatory carboxypeptidase B or corresponding procarboxypeptidase B from insulinogen or agent or agent box to test protein and diagnose pancreatic disease.

Description

technical field [0001] The invention relates to the field of molecular biology, and mainly relates to a method for improving the performance of gene expression products through site-directed mutation. Specifically, it is a method of improving the activity of the mutant carboxypeptidase B expression product by selecting a specific cysteine ​​site or other gene sites that affect the formation of disulfide bonds. Background technique [0002] Pancreatic carboxypeptidase B (carboxypeptidase B, CPB, EC 3.4.17.2) was discovered in 1958 (see Folk JE, Gladner JA, Carboxypeptidase B J Biol Chem 1958; 231:379-391). It is a digestive enzyme whose levels are barely detectable in normal serum. Active CPB is found in serum only during pancreatitis and its plasma half-life is several minutes, probably due to lack of glycosylation. [0003] Carboxypeptidase B is a zinc-containing exopeptidase in which zinc ions are present as a cofactor necessary for enzymatic activity. Carboxypeptidase ...

Claims

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Application Information

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IPC IPC(8): C12N9/48C12N15/57C12N15/63C12N1/21C12P21/06C12Q1/37G01N33/68G01N33/573
Inventor 李素霞王福清袁勤生
Owner BEIJING GUANHONG TECH
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