Production and application of high stability recombinant trypsin

A trypsin and trypsinogen technology, which is applied in the production and application fields of recombinant trypsin, can solve the problems of low renaturation rate of inclusion bodies, inability to obtain effective expression of trypsin, and low yield of recombinant trypsin, etc.

Active Publication Date: 2011-02-09
上海雅心生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Expressing trypsin alone cannot achieve effective expression, because the existence of a small amount of active trypsin can cause toxicity to the expression host cell. Some people express it in the form of trypsinogen. Compared with trypsin, the N-terminus of trypsinogen is only 7 more. a

Method used

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  • Production and application of high stability recombinant trypsin
  • Production and application of high stability recombinant trypsin
  • Production and application of high stability recombinant trypsin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1, the soluble expression of fusion protein (N-terminal 159 amino acids+human trypsinogen)

[0069] A polypeptide consisting of 159 amino acids is added to the N-terminus of human trypsinogen (GenBank: M27602.1, positions 17-247 (ie, excluding the signal peptide before the leader peptide), its sequence is (SEQ ID NO: 1 ):

[0070] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVRGSGMKETAAAKFERQHMDSPDLGTENLYFQS

[0071] Among them, there are 12 aspartic acid (D).

[0072] Design the following primers:

[0073] Forward: aa ggatcc AAAATCGTGGGTGGTTAC (SEQ ID NO: 6);

[0074] Reverse: cc AAGCTT TTAAGAGTTAGCAGCGA (SEQ ID NO: 7);

[0075] Using the human pancreas cDNA library as a template, the coding sequence of human trypsinogen was obtained by performing PCR amplification with the aforementioned primers.

[0076] The sequence obtained above was digested with HindIII / BamHI and ...

Embodiment 2

[0079] The purification of the fusion protein that embodiment 2, embodiment 1 obtain

[0080] The supernatant collected after the above-mentioned induced expression cells were sonicated was used as a sample solution to purify the recombinant fusion protein.

[0081] Use 2×15cm chromatography column, the ion exchange column is equilibrated with 20mM Tris-HCl, pH 8.0 buffer in advance, then the supernatant is loaded, equilibrated with the same buffer until the baseline is stable, then use 0-1M NaCl or 0~0.5M CaCl 2 Gradient elution, collection in sections, measure the OD280 and enzyme activity of each tube, combine the collected solutions with enzyme activity, and measure the total enzyme activity and total protein content. Combine the collection tubes with high enzyme activity to obtain purified active recombinant trypsin.

[0082] The collected purified solution was tested by SDS-PAGE for the purification results, see figure 2 . Among them, lane 1 is the loading solution;...

Embodiment 3

[0084] Embodiment 3, activation conditions

[0085] Using porcine pancreatic trypsin (purchased from Sigma Company), the fusion protein obtained from the aforementioned purification was activated by enzymatic hydrolysis to obtain active recombinant trypsin. Wherein, the amount of porcine pancreatic trypsin added is: porcine pancreatic trypsin:fusion protein (w:w)=1:1000. After activation, the recombinant trypsin with the activity of the amino acid fused at the N-terminus and the 7 amino acids at the N-terminus removed was obtained.

[0086] The enzymatic hydrolysis conditions are shown in Table 1.

[0087] image 3 It is the electrophoresis identification result of the enzymolysis solution obtained after enzymolysis at 4°C for 25 hours. Among them, swimming lane 1 is the electrophoresis result of the purified enzyme solution (fusion protein); swimming lane 2 is the corresponding figure 2 Lane 3 is the electrophoresis result after enzymolysis; lane 3 is the electrophoresis...

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Abstract

The invention relates to production and application of high stability recombinant trypsin, and discloses a method for producing the recombinant trypsin, which comprises the following steps of: (1) performing recombinant expression on a fusion protein, wherein the fusion protein sequentially comprises 40-200 amino acid sequences and trypsinogen sequences from an N terminal to a C terminal, and is solubly expressed; and (2) cutting off the 40-200 amino acid sequences and a leader sequence at the N terminal of the fusion protein to obtain the recombinant trypsin. The method realizes the soluble expression of the trypsin, and the high activity and high stability trypsin can be obtained.

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 trypsin. Background technique [0002] Trypsin (Trypsin, EC3.4.21.4) is synthesized in the pancreas in the form of trypsinogen, an enzyme precursor, and is secreted as a component of pancreatic juice. Active trypsin is an endopeptidase that can cut off the carboxyl side of lysine and arginine residues in the polypeptide chain. It not only acts as a digestive enzyme, but also restricts and decomposes the precursors of other enzymes such as chymotrypsinogen, carboxypeptidase, and phospholipase, and activates it. It is the most specific protease and is important in protein sequencing. tool enzyme. [0003] It is precisely because trypsin is a highly active endopeptidase that can hydrolyze any peptide bond formed by the carboxyl terminus of lysine and arginine residues exposed on the surface of the...

Claims

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Application Information

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IPC IPC(8): C12N9/76C12R1/19C12N15/70C12N9/96C12N15/62
Inventor 冯矗赵致
Owner 上海雅心生物技术有限公司
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