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Production and application of high-stability recombination carboxypeptidase B

A carboxypeptidase and recombinant protein technology, which is applied in the production and application fields of recombinant carboxypeptidase B, and can solve the problem that proteins cannot spontaneously complete the folding process and the like

Active Publication Date: 2012-11-14
上海雅心生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In some cases, without the presence of IMC, the protein cannot complete the folding process spontaneously. This protein folding phenomenon dependent on the leader sequence was first discovered by Ikemura et al. in the study of subtilisin in 1987.

Method used

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  • Production and application of high-stability recombination carboxypeptidase B
  • Production and application of high-stability recombination carboxypeptidase B
  • Production and application of high-stability recombination carboxypeptidase B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1, the recombinant expression of carboxypeptidase B

[0069] 1. Recombinant expression of carboxypeptidase B (procarboxypeptidase B) with a leader sequence

[0070] Design the following primers:

[0071] Forward: 5'GCG CCA TGG CAT GCT TCC GAG GAG CAC TTT GATGGC-3' (SEQ ID NO: 3), containing Nco I restriction enzyme site;

[0072] Reverse: 5'-CGC AAG CTT TCA CTA ATA TAG ATG TTC TCG GAC ATAATT-3' (SEQ ID NO: 4), containing a Hind III restriction enzyme site and a stop codon.

[0073] Using the pancreatic cDNA library (purchased from Invitrogen) as a template, the aforementioned primers were used to perform PCR amplification to obtain the coding sequence of carboxypeptidase B with a leader sequence.

[0074] The sequence obtained above was digested with Nco I / HindIII and inserted into the corresponding site of the pET expression vector (purchased from Invitrogen), and the correctly inserted recombinant expression vector was identified by sequencing. The...

Embodiment 2

[0090] Example 2, the role of the leader sequence as an intermolecular chaperone on the folding of carboxypeptidase B

[0091] 1mg / ml purified recombinant carboxypeptidase B was denatured in 8M urea for 2h, and directly diluted 10-fold into refolding buffer (100mM Gly-NaOH, pH 9.5, GSH 1mM, 0.1mM ZnCl 2 ), the final protein concentration in the renaturation buffer is 0.1 mg / ml. Start timing from dilution and renaturation, and sample 0.1ml at regular intervals to measure activity. 100% enzyme activity is defined as the activity of the original enzyme solution (1 mg / ml) diluted 10 times directly at pH 9.5 with 0.1MGly-NaOH.

[0092] The activity assay uses hippuryl-L-arginine as the substrate, and the activity unit is defined as the percentage of substrate hydrolysis per minute at a substrate concentration of 0.001M. The activity buffer is: 0.025M Tris-HCl, pH7. 65 with 0.1M NaCl.

[0093] According to the molar ratio of CPB:pro=1:1, the leader sequence (pro) was added to the...

Embodiment 3

[0095] Example 3, the refolding effect of the leader sequence as an intramolecular chaperone of CPB on carboxypeptidase B

[0096] 0.5 mg / ml of the refolded and purified zymogen proCPB (recombinant carboxypeptidase B with a leader sequence, obtained after refolding without trypsin enzymatic activation and purified) was denatured in 8M urea for 24 hours, and directly Dilute 5 times into the refolding buffer, refold at room temperature, and the final protein concentration at this time is 0.1mg / ml. Start timing from dilution and renaturation, take 1ml at intervals, immediately add trypsin for enzymatic activation, wherein the mass ratio of procarboxypeptidase B: trypsin is 10:1, and measure CPB activity after incubation at 37°C for 40min. 100% enzyme activity is defined as the activity determined after the original enzyme solution (1 mg / ml) is diluted 5 times and activated by enzymatic hydrolysis in the same method.

[0097] The zymogen in the form of inclusion body (that is, th...

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Abstract

The invention relates to production and application of high-stability recombination carboxypeptidase B. The invention also discloses a method for producing recombination carboxypeptidase B, which comprises the following steps: (1) carrying out recombinant expression on carboxypeptidase B with a mutated leading sequence at the N terminus to obtain a recombination protein in an inclusion body form;(2) denaturing and renaturing the recombination protein in an inclusion body form to obtain a soluble recombination protein; and (3) removing leading sequences of the carboxypeptidase B with a leading sequence at the N terminus of the soluble recombination protein to obtain the recombination carboxypeptidase B. The invention uses an intramolecular chaperone to enhance the in vitro refolding of the carboxypeptidase B, and the obtained recombination carboxypeptidase B has high stability and can well resist the enzymolysis of trypsase.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to the production and application of a highly stable recombinant carboxypeptidase B. Background technique [0002] The content of carboxypeptidase B in the pancreas is small, and the carboxypeptidase B extracted from the pancreas cannot guarantee the removal of other protease activities. The production of recombinant carboxypeptidase B solves this problem, but in the production process of recombinant carboxypeptidase B, Especially in the production process of Escherichia coli, active carboxypeptidase B can be obtained through further denaturation and renaturation in the form of inclusion bodies, but its renaturation rate is the bottleneck affecting its large-scale production. [0003] Many prokaryotic and eukaryotic proteases are synthesized in the form of zymogens. After folding and maturation, the leader sequence is recognized, excised and degraded by the co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/48C12N15/70C07K14/47C12N15/12C12R1/19
Inventor 冯矗赵致
Owner 上海雅心生物技术有限公司
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