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Recombinant bovine enterokinase, preparation method and use thereof

A bovine enterokinase and heavy chain technology, applied in the biological field, can solve problems such as low protein activity and difficult renaturation of inclusion body products

Active Publication Date: 2009-03-18
SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] Although a lot of research has been done on recombinant enterokinase, the inclusion body product of enterokinase light chain obtained from the widely used Escherichia coli expression system has problems of difficult renaturation and low protein activity. With the development of technology, there is still a need for recombinant enterokinase with high expression, good activity and suitable for production

Method used

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  • Recombinant bovine enterokinase, preparation method and use thereof
  • Recombinant bovine enterokinase, preparation method and use thereof
  • Recombinant bovine enterokinase, preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Cloning of bovine EK cDNA fragment

[0039] Take 150 mg fresh bovine duodenum, extract total RNA with Trizol reagent (product of Invitrogen), take 0.5 μl total RNA, add 3 μl oligo(dT)18 primer, add water to a total volume of 10.5, and mix well. Place at room temperature for 10 minutes, and centrifuge at high speed for 5 minutes. Then add 4.0 μl 5×reverse transcription reaction buffer, 0.5 μl RNase inhibitor, 2.0 μl 10mMdNTP, 2.0 μl DTT, 1 μl MMLV reverse transcriptase, mix well, and react at 37°C for 120min. Take 1.5 μl RT product, add 100 pmol upstream primer (5'-aagcttatggggtcaaagcgaagtgt-3') and downstream primer (5'-gaattctcaatgtagaaaactttgtatcc-3', design primer with reference to the coding sequence of bovine Enterokinase in CenBank, sequence number: U09859), 2.5 μl 10 ×PCR buffer, 2μl 2.5mMd NTP, 2.5DMSO, 0.25Taq enzyme, add water to a final volume of 25μl, mix well and quickly add 5μl paraffin oil for PCR amplification. The reaction conditions were dena...

Embodiment 2

[0040] Example 2 Design of EKLm and construction of pET39b-EKL and pET39b-EKLm expression plasmids

[0041] In order to improve the activity of enterokinase, the inventors hereby refer to bovine enterokinase light chain and its inhibitor VD 4 The crystal structure of the complex of K-chloroform (Code no: 1EKB, Protein Dtabase Bank), observed by VD 4 K is the center 5 In the case of amino acid residues within the range, select the residues that may enhance the binding activity of EKL and DDDDK after mutation, and mutate it into R to enhance its binding activity with VD 4 The binding force of D in K. A total of 9 mutants were designed, such as figure 1 shown.

[0042] Using the correctly identified pMD18-T-EK as a template, the cDNA fragment of the light chain coding region of bovine enterokinase was amplified by PCR. The upstream primer has an EcoRI restriction site and an enterokinase recognition site, and the downstream primer has a HindIII restriction site and a stop c...

Embodiment 3

[0043] Example 3 Induced expression of pET39b-EKL and pET39b-EKLm

[0044] The thioredoxin analog DsbA provided on the pET39b vector can help the protein to fold normally, thereby producing an active soluble protein. Each engineered bacteria was inserted into LB medium. Place in a shaker at 37°C and 250rpm for overnight culture. The overnight seed solution was added to the 2YT medium, and placed in a shaker for cultivation at 37° C. and 250 rpm. When OD 600 When the temperature is 0.6, 0.1 mM IPTG is added to induce, and placed in a shaker to continue culturing at 30°C and 250 rpm. After 2 hours of induction, samples were taken, centrifuged to remove the supernatant, and the precipitate was placed in the refrigerator. Then samples were taken every 1 hour, and the supernatant was also centrifuged to remove, and the precipitate was placed in the refrigerator. After 6 hours of induction, the bottle was closed, and the supernatant was removed by centrifugation. The induced ex...

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Abstract

The invention discloses fusion protein comprising a recombined bovine enterokinase light chain mutant and a recombined bovine enterokinase light chain mutant with a heavy chain structural domain, wherein the heavy chain structural domain is connected with the light chain mutant through a short peptide. The invention further discloses amino acid sequences of the mutants and the fusion protein, a method for preparing the same, and applications of the mutants and the fusion protein as fusion protein cutting agents. The fusion protein comprising the recombined bovine enterokinase light chain mutant and the recombined bovine enterokinase light chain mutant with the heavy chain structural domain can be obtained through simple steps of separation and purification and have the advantage of high activity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention discloses a recombinant bovine enterokinase mutant and a recombinant bovine enterokinase fusion protein, a preparation method and an application thereof. Background technique [0002] Enterokinase (Enterokinase, EK or Enteropeptidase, EP, EC3.4.21.9) is a heterodimeric serine protease present in the mammalian duodenum. The molecular mass is 150kDa, and it consists of one heavy chain of 115kDa and one light chain of 35kDa. It specifically hydrolyzes protein substrates at a pH value of 4.5-9.5 and a temperature range of 4-45°C. Since the target polypeptide released by cleavage of the fusion protein by EK has the same N-terminal amino acid sequence as the wild type, it can be used as a cleavage reagent for the fusion protein in the protein expression system. However, the source of natural enterokinase is limited after all, and the cost of extraction and separation i...

Claims

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Application Information

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IPC IPC(8): C12N9/64C12N15/57C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C07K19/00C12N15/62C12N15/11
Inventor 郭亚军侯盛谈珉王皓李博华张大鹏钱卫珠
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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