Preparation method of recombinant carboxypeptidase B

A technology of carboxypeptidase and enzyme cleavage site, which is applied in the field of preparation of recombinant carboxypeptidase B, and can solve problems such as the risk of pharmaceutical proteins

Inactive Publication Date: 2012-07-04
JIANGSU WANBANG BIOPHARMLS
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  • Application Information

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Problems solved by technology

[0006] These three carboxypeptidase B genes are all animal genes such as pigs and rats, and are not real human carboxypeptidase B genes. When they are used in the production of recombinant pharmaceutical proteins (such as recombinant human insulin, etc.), they may appear in the final product. Some unknown exogenous virus or exogenous protein sequences are brought in, and there is a certain risk for medicinal proteins

Method used

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  • Preparation method of recombinant carboxypeptidase B
  • Preparation method of recombinant carboxypeptidase B
  • Preparation method of recombinant carboxypeptidase B

Examples

Experimental program
Comparison scheme
Effect test

example 3

[0053] Example 3 Carboxypeptidase B Purification

[0054] After the fermentation, the cells were collected by centrifugation, and the crushing buffer (25mmol / L Tis-HCL+5mmol / L EDTA, pH7.5) was added at a weight-to-volume ratio of 1:10. The inclusion body precipitate was collected, and the weight loss of the inclusion body was 30g / L fermentation broth. Add the precipitate to washing buffer (2 mol / L urea + 1.5% Triton) at a weight-to-volume ratio of 1:10, stir magnetically at room temperature for 1 hour, and wash the precipitate collected by centrifugation twice with washing buffer. The inclusion body was then dissolved overnight with inclusion body dissolution buffer (8mol / L urea+10mmol / LEDTA+25mmol / L Tis-HCL, pH7.5) at a weight-to-volume ratio of 1:10. The dissolved inclusion bodies were centrifuged and ultrafiltered to remove impurities (such as Figure 4 shown), diluted to a protein concentration of 0.2mg / ml, in refolding buffer (0.2mol / L urea+10mmol / LEDTA+25mmol / L Tis-HCL...

example 4

[0055] The activity measurement of example 4 carboxypeptidase

[0056] The activity of carboxypeptidase B was determined by hydrolyzing hippuronyl-L-arginine at 25°C and pH 7.65,

[0057] Buffer: 25 mM Tris HCl buffer containing 100 mM NaCl at 25°C, pH 7.65.

[0058] Substrate solution: 1.0 mM hippurinyl-L-arginine solution.

[0059] Carboxypeptidase B enzyme solution: (prepared when used, containing 4-8 enzyme activity units / ml water)

[0060] Record the increase in absorption value within 5 minutes ( Figure 6 ), the maximum absorbance / time is obtained using the maximum linear velocity of the assay and blank.

[0061]

[0062] df: dilution factor

[0063] It was determined that the purified recombinant carboxypeptidase B had an activity of 98 units per milligram.

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

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Abstract

The invention relates to a preparation method of recombinant carboxypeptidase B. The preparation method comprises the following steps of: firstly introducing plasmids with recombinant carboxypeptidase B protogene sequence into Escherichia coli cells to construct a recombinant engineering strain, fermenting for inducing and expressing recombinant carboxypeptidase B protogene, and carrying out renaturation, pancreatin conversion as well as separation and purification to obtain carboxypeptidase B with activity. The preparation method provided by the invention has the characteristic that the expressed carboxypeptidase B gene has completely identical sequence with human carboxypeptidase B.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a preparation method of recombinant carboxypeptidase B. technical background [0002] Carboxypeptidase B (EC3.4.17.2, carboxypeptidase B, CPB) is an exopeptidase that can specifically hydrolyze C-terminal basic amino acids (Arg, Lys) of proteins or polypeptides. Carboxypeptidase B is widely used in the fields of protein sequencing, pancreatitis diagnosis and recombinant drug protein production. Currently commercially available carboxypeptidase B mainly comes from three sources: [0003] The first is natural carboxypeptidase B, such as obtained by extracting from the pancreas of animals such as pigs and cattle (such as Li Xianlin et al. "A preparation method of carboxypeptidase B and its composition": Chinese Invention Patent Publication CN200510030397.X) , the porcine pancreas was treated with acetone to make acetone powder, and then through the steps of salting out, hydropho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N1/21C12N9/48C12N15/57C12R1/19
Inventor 文良柱温传彬王伟刚
Owner JIANGSU WANBANG BIOPHARMLS
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