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Non-self-cutting trypsin and preparation method thereof

A technology of trypsin and porcine trypsin, applied in the direction of biochemical equipment and methods, enzymes, peptidases, etc., can solve problems such as instability

Active Publication Date: 2019-08-06
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

However, due to the self-cleavage of trypsin itself under enzyme cleavage conditions, it is extremely unstable

Method used

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  • Non-self-cutting trypsin and preparation method thereof
  • Non-self-cutting trypsin and preparation method thereof
  • Non-self-cutting trypsin and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Embodiment 1, the mutation analysis of porcine trypsin (Pt)

[0085] Porcine trypsin (Pt) is prone to autodegradation. Its amino acid sequence is as follows (SEQ ID NO: 1):

[0086] IVGGYTCAANSIPYQVSLNSGSHFCGGSLINSQWVVVSAAHCYKSRIQV LGEHNIDVLEGNEQFINAAKIITHPNFNGNTLDNDIMLIKLSSPATLNS VATVSLP CAAAGTECLISGWGNTKSSGSSYPSLLQCLKAPVLSDSSCKSSYPGQITGNMICVGFLEGGKDSCQGDSGGPVVCNGQLQGIVSWGYGCAQKNKPGVYTKVCNYVNWIQQTIAAN

[0087] The present inventors studied the stability of wild-type Pt, and analyzed its self-degrading short peptide, and found its most important self-degrading fragment. The amino acid positions corresponding to these self-degrading fragments are Arg49, Arg99, and Arg107, respectively. Among them, Arg49 and Arg107 may be the secondary degradation sites of Pt.

[0088] The present inventors used the method of site-directed mutagenesis to study the effect of the mutation at position 99 on the stability, including Pt-R99L (mutation of Arg to Leu) and Pt-R99H (mutat...

Embodiment 2

[0091] Embodiment 2, the effect of Arg99 site on stability

[0092] In Example 1, the inventors designed and recombinantly expressed rPt, rPt-R99L (PTL), rPt-R99H (PTH), purified them, and studied their stability.

[0093] 1. Temperature stability

[0094] The inventors compared the stability of rPt, rPt-R99L, and rPt-R99H at 60°C. The enzymes were preserved in 2mM hydrochloric acid and then incubated in a 60°C water bath, and the activity was measured at regular intervals.

[0095] The result is as Figure 1A shown. It can be seen from the figure that the activities of the three decreased the fastest in the first 2 hours, rPt decreased by 35%, rPt-R99L and rPt-R99H decreased by 20% and 18% respectively, and the activity of the three decreased slowly after 2 hours, but they could It is obvious that the overall activity of rPt-R99L and rPt-R99H is 15%-20% higher than that of wild-type rPt, and the thermal stability of rPt-R99L is about 5% higher than that of rPt-R99H.

[009...

Embodiment 3

[0107] The stability of the mutant further mutated on the basis of embodiment 3, rPt-R99H

[0108] 1. Temperature stability

[0109] In the present embodiment, porcine trypsin (rPt) and mutant thereof are divided into following three kinds:

[0110] PTH: rPt-R99H;

[0111] PTGH: rPt-R49G-R99H;

[0112] PTHT: rPt-R99H-R107T;

[0113] PTGHT: rPT-triple mutation, ie rPt-R49G-R99H-R107T.

[0114] Synthesize the above enzyme mutants, dilute the purified above four enzymes to 0.5mg / ml, then incubate them in a water bath at 60°C, measure their activity at 0h, 2h, 4h, 8h, and 16h, and take the activity at 0h As 100%, calculate the activity residual rate at other times, and make a graph to show the relationship between time and activity residual rate.

[0115] The result is as figure 2, Comparing the stability of the four mutants at 60°C, it can be seen that the rPt-R49G-R99H, rPt-R99H-R107T, and rPT-triple mutant mutants constructed on the basis of rPt-R99H are rPt-R99H, rPt-R9...

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Abstract

The invention relates to a non-self-cutting trypsin and preparation method thereof. The invention provides a trypsin mutant, and compared with a wild type, the stability of the mutant is better. The present invention also provides a method of methylation modification of the trypsin mutant, so that the trypsin effectively avoids the generation of self-cutting.

Description

technical field [0001] The invention belongs to the field of bioengineering, and more specifically, the invention relates to a trypsin without self-cutting and a preparation method thereof. Background technique [0002] Trypsin can recognize and hydrolyze arginine and lysine with high specificity, and is currently widely used in the preparation of biological products such as insulin and chondroitin sulfate collagen. It has wide application value in medicine, light industry and scientific research. Trypsin can be widely used in the pharmaceutical industry as a proteolytic enzyme drug. [0003] Trypsinogen can be activated into single-chain β-type trypsin by hydrolyzing the N-terminal zymogen propeptide. Next, several multi-chain trypsin α-type trypsins connected by disulfide bonds can be produced by self-hydrolysis. Different activities and thermal stability are the main differences in their properties. β-type trypsin is relatively higher in enzymatic activity than α-type ...

Claims

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Application Information

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IPC IPC(8): C12N9/76G01N33/68
CPCC12N9/6427C12Y304/21004G01N33/6854G01N33/6848
Inventor 赵致王之可刘晓
Owner 上海雅心生物技术有限公司
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