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Method for selectively protecting restriction enzyme cutting sites in trypsin digestion process

A trypsin and selective technology, applied in the field of enzyme engineering, can solve the problems of target protein activity reduction and inactivation, and achieve the effect of reducing affinity and eliminating enzyme cutting effect

Active Publication Date: 2020-09-01
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the position of arginine in the amino acid sequence is close to the active center site of the protein itself, or itself is a key residue for its activity, the mutation may lead to reduced or even inactivated activity of the target protein

Method used

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  • Method for selectively protecting restriction enzyme cutting sites in trypsin digestion process
  • Method for selectively protecting restriction enzyme cutting sites in trypsin digestion process
  • Method for selectively protecting restriction enzyme cutting sites in trypsin digestion process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: A method for selectively protecting the enzyme cleavage site during trypsin digestion

[0021] A method for selectively protecting the enzyme cleavage site during trypsin digestion, comprising a polypeptide to be digested by trypsin, the structure of which is: polypeptide 1: SOD fragment 1 + arginine + GLP1 sequence + GCGGGGGG + SOD Fragment 2.

[0022] Among them, the sequence of SOD fragment 1 is:

[0023] ATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGP;

[0024] The sequence of GLP1 (7-37aa) is: HAEGTFTSDVSSYLEGQAAKEFIAWLCKGRG, in which the V at position 33 is mutated to C, in order to cross-link with C in GCGGGGGG to form a disulfide bond. The C-terminus of wild-type GLP1 does not have the sequence GCGGGGGG, which is connected to the back when designing the gene, which can be called a linker.

[0025] SOD fragment 2 sequence is: MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEG.

[0026] The arginine of the polypeptide is flanked by cysteines,...

Embodiment 2

[0038] Example 2: A method for selectively protecting the enzyme cleavage site during trypsin digestion

[0039] Polypeptide 3: SOD protein fragment 1 + arginine + GLP1 sequence + GCGGGGGG + SOD fragment 2

[0040] The sequence of SOD fragment 1 is ATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGP;

[0041] The GLP1 sequence is (7-37aa)HAEGTFTSDVSCYLEGQAAKEFIAWLVKGRGC. Among them, the S at the 18th position is mutated to a C, and a C is added after the G at the 37th position.

[0042] SOD fragment 2 sequence is MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEG.

[0043] Peptide triple sequence such as Figure 6 shown.

[0044] Check whether the arginine in polypeptide 1, polypeptide 2 and polypeptide 3 can be digested by trypsin according to the following steps.

[0045] Synthetic coding gene: Synthesize the target gene sequence through the gene synthesis company Hefei Jixiang Biotechnology Co., Ltd. The sequence is as follows:

[0046] Sequence 1:

[0047]CATATG...

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PUM

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Abstract

A method for selectively protecting restriction enzyme cutting sites in a trypsin digestion process is characterized in that a polypeptide to be digested by trypsin is prepared, cysteines are arrangedon the two sides of arginine of the polypeptide, two cysteines form a disulfide bond, and the disulfide bond stretches across arginine. Appropriate sites are selected to insert cysteine or unimportant amino acids are mutated into cysteine on two sides of arginine needing to be protected in polypeptide, and two cysteines stretch over arginine to be protected to form a disulfide bond. Through the conformation change caused by the disulfide bond, steric hindrance is formed at the enzyme cutting sites, the affinity of trypsin and arginine carboxyl terminals is reduced, and the enzyme digestion effect is greatly weakened or even eliminated.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and relates to a method for selectively protecting enzyme cutting sites during trypsin digestion. Background technique [0002] Trypsin is an endopeptidase, and its cleavage site is / K / R-\P, which can cleave the C-terminus of arginine and lysine in the amino acid sequence of the protein (such as arginine / lysine Amino acid followed by proline will not be cleaved). In the enzyme cleavage reaction, in order to prevent the lysine in the peptide chain from being cleaved by trypsin, citraconic anhydride or maleic anhydride is usually used to react with the ε amino group on the lysine side chain to protect the C-terminus of lysine from Digested by trypsin. However, there is no good protection method for arginine in the peptide chain, and arginine can only be mutated into other amino acids. If the position of arginine in the amino acid sequence is close to the active center site of the prot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/02
CPCC12N9/0089C07K14/605C12Y115/01001C07K2319/50C07K2319/31Y02P20/55
Inventor 王鹏孙磊郑之明吕荟王丽王晗赵根海
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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