A kind of low-temperature matrix metalloproteinase and its coding gene and application

A technology of matrix metals and coding genes, applied in the field of enzyme engineering, can solve the problems of lack of oxygen supply, damage to cell culture activity, etc., and achieve the effect of high-efficiency protein degradation activity

Active Publication Date: 2021-04-02
TECHNICAL INST OF PHYSICS & CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Take collagenase as an example for the digestion of tissue cells in the early stage of mammalian cell culture. If the digestion process is carried out at 37°C, the animal cells are in an active state, and the lack of effective oxygen supply during the reaction causes the cells to undergo anaerobic respiration and produce a large amount of And accumulate metabolic by-products such as lactic acid, which impairs the activity of subsequent cell culture processes

Method used

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  • A kind of low-temperature matrix metalloproteinase and its coding gene and application
  • A kind of low-temperature matrix metalloproteinase and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Obtaining of low-temperature matrix metalloproteinase gene and expression of low-temperature matrix metalloproteinase 1. Establishment of low-temperature matrix metalloproteinase gene transcription library

[0036] The adult Antarctic fish of Roche (Notothenia rossii) was dissected by vivisection, and the mouth samples were taken, which were quick-frozen in liquid nitrogen and stored at -80°C for later use. After tissue samples were ground with liquid nitrogen, total RNA was extracted with RNAiso Plus (Takara, Dalian) kit. RNA samples were digested with RNase-free DNase I to remove genomic DNA contamination, then detected by agarose gel electrophoresis and UV spectrophotometer, and the concentration was adjusted to 500ng / μL. Using the above RNA sample as a template, it was reverse-transcribed into cDNA by AMV reverse transcription kit (Promega), and stored at -20°C.

[0037] 2. Acquisition of low temperature matrix metalloproteinase gene

[0038] Using the c...

Embodiment 2

[0047] Example 2 Activity Detection of Low Temperature Matrix Metalloproteinases (Hydroxyproline Detection Method)

[0048] 2.1 Test principle

[0049] Matrix metalloproteinase catalyzes the decomposition of solid insoluble collagen into soluble peptides and enters the solution. After the reaction is terminated, unreacted proteins are filtered out, and the filtrate is heated and acidified to free amino acids. Hydroxyproline is a specific amino acid of collagen, and the quality of free hydroxyproline in the acid hydrolysis solution is measured by the hydroxyproline colorimetric method, which indirectly indicates the amount of hydrolyzed collagen. The ratio of this value to the total content of hydroxyproline in the substrate collagen is called the degradation rate of collagen, which can reflect the collagen hydrolysis activity of matrix metalloproteinases.

[0050] 2.2 Experimental method

[0051] With Tris-HCl buffer (pH=7.4, containing 5mmol / ml CaCl 2 , 0.5mmol acetic acid...

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Abstract

The invention discloses low-temperature matrix metalloprotease, and a coding gene and application thereof. The low-temperature matrix metalloprotease disclosed in the invention is a protein as described in (a) or (b): (a) a protein composed of an amino acid sequence as shown in SEQ ID No. 1 in a sequence table; and (b) a protein having protein degradation activity at low temperature and prepared by subjecting the amino acid sequence as shown in SEQ ID No. 1 in the sequence table to substitution and / or deletion and / or insertion of one or more amino acid residues. The invention further disclosesapplication of the low-temperature matrix metalloprotease to degradation of protein under low temperature conditions. The low-temperature matrix metalloprotease has efficient protein degradation activity at a low temperature of 10 to 20 DEG C and has low thermal inactivation temperature.

Description

technical field [0001] The present invention relates to the field of enzyme engineering. More specifically, it relates to a low-temperature matrix metalloproteinase and its coding gene and application. Background technique [0002] Matrix metalloproteinases, are a large class that require Ca 2+ , Zn 2+ And other metal ions as cofactors, and proteases that can degrade various extracellular matrix components (collagen, gelatin, viscous protein, fibronectin, proteoglycan, etc.). One of the matrix metalloproteinases with collagen degrading activity is widely used in light industry production, medical and health industries and other industries. [0003] Collagenase can be used as a dyeing auxiliary agent for leather, because it has a certain catalytic activity on the collagen fiber matrix, thereby improving the dyeing performance of leather. Leather is a material composed of collagen. After the collagen is tanned with various cross-linking agents (such as chromium), the struc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64C12N15/57
CPCC12N9/6491
Inventor 郭燕川邵明非
Owner TECHNICAL INST OF PHYSICS & CHEMISTRY - CHINESE ACAD OF SCI
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