Preparation method of active carboxypeptidase B

A carboxypeptidase, active technology, applied in the field of preparation of active carboxypeptidase B, can solve problems such as complicated steps and the like

Inactive Publication Date: 2007-07-04
TIBET JINKE GRP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for preparing active CPB by directly expressing CPB, so as to overcome the defects of complicated steps in the prior art

Method used

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  • Preparation method of active carboxypeptidase B
  • Preparation method of active carboxypeptidase B
  • Preparation method of active carboxypeptidase B

Examples

Experimental program
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Effect test

Embodiment 1

[0044] From the LB solid culture plate (LB culture solution, add agar powder to a concentration of 1.5 (wt%)) to pick a recombinant single colony in 5ml LB liquid culture solution containing 100 μg / ml ampicillin (1% (wt%) tryptone, 0.5 %(wt%)yeast extract, 0.5%(wt%)NaCl, pH7.0), 37°C, shake the bacteria overnight; the overnight bacteria were inoculated at 1% (v / v) in 250ml LB containing 100μg / ml ampicillin culture medium at 37°C until OD 600 0.5, 0.5mmol / L IPTG induction, lower the temperature to 18°C, continue to cultivate for 5 hours, harvest the bacteria, centrifuge and discard the supernatant, suspend the bacteria sediment with 5-100mmol / L pH8.0 TE buffer, ultrasonically break the bacteria body, centrifuged to take the supernatant, and measured the enzyme activity in the supernatant, the activity was 0.15u / ml, adding ZnCl 2 Make the final concentration of 0.01mmol / L in the supernatant, shake gently and mix well, and after standing for 30min, measure the activity of CPB, w...

Embodiment 2

[0047] From the LB solid culture plate (LB nutrient solution, add agar powder to a concentration of 1.5 (wt%)) to pick a recombinant single colony in 5ml LB liquid nutrient solution containing 100 μg / ml ampicillin (1% (wt %) tryptone, 0.5% (wt%) yeast extract, 0.5% (wt%) NaCl, pH 7.0), 37 ° C, shake the bacteria overnight; the overnight bacteria were inoculated at 1% (v / v) in 250ml LB containing 100μg / ml ampicillin resistant In culture medium, culture at 37°C to OD 600 0.5, 0.5mmol / L IPTG induction, 1mmol / L IPTG induction, after 37 ℃ continued culture for 3 hours, harvest the bacteria, centrifuge to discard the supernatant, suspend the bacteria pellet with 20mmol / L pH8.0 TE buffer, and ultrasonically break Bacteria were centrifuged, and the precipitate was collected. It was identified by SDS-PAGE electrophoresis that the precipitate was the expression part of the inclusion body of CPB. The inclusion body was collected, suspended with 1% (v / v) Triton-X 100, fully washed, centri...

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Abstract

The invention relates to a method for preparing active carboxypeptidase B. It comprises following steps: expressing the recombination bacteria carrying with CPB in the form ofinclusion bodies; and separating and getting active CPB. It is characterized in that the plasmid of said recombiantino bacteria is pT7- 473- CPB, and the host is E coli Bl21 (DE3). The invention realizes the active CPB preparation by direct expression of CPB, and overcomes complicate steps in current methods.

Description

technical field [0001] The invention relates to a preparation method of active carboxypeptidase B. Background technique [0002] Carboxypeptidase B (carboxypeptidase B, referred to as CPB,) is a zinc-containing pancreatic exopeptidase. CPB contains 307 amino acids with a molecular mass of 35ku. Since CPB specifically hydrolyzes the basic amino acids (arginine, lysine or ornithine) at the C-terminal of the peptide chain, it can be used for sequence analysis of proteins and polypeptides, especially for determining the carboxy-terminal amino acids. In addition, the main commercial use of CPB at present is as an industrial enzyme applied to the industrial production of insulin. [0003] Currently, the methods for preparing active CPB can be divided into three categories. One is to extract and activate the active CPB from animal pancreas. The defect of this method is that the application of CPB is limited because the obtained CPB may contain other substances (such as viruses o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/48C12N1/21
Inventor 李素霞袁勤生
Owner TIBET JINKE GRP
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