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Glutamine transaminase zymogen gene for streptomyces hygroscopicus and expression thereof

A technology of transglutaminase proenzyme and transglutaminase enzyme, which is applied in the field of transglutaminase proenzyme and can solve the problem of low enzyme activity and the like

Inactive Publication Date: 2008-02-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Christian K et al. (Christian K.et, al.Enzyme and Microbial Technology2007, 40:1543-1550) used the transglutaminase gene of Streptomyces mobara to obtain partial soluble expression in Escherichia coli, but the enzyme activity was not high

Method used

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  • Glutamine transaminase zymogen gene for streptomyces hygroscopicus and expression thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: Obtaining of the transglutaminase gene of Streptomyces hygroscopicus

[0052] The complete amino acid sequence of transglutaminase from Streptomyces platensis, Streptomyces mobaraensis, Streptomyces cinnamoneus and Streptomyces fradiae was found through the NCBI gene bank. According to the results reported in the literature, the starting sequence and termination sequence of the zymogen were found, and then the corresponding base sequences were compared; on the other hand, after the zymogen from natural Streptomyces hygroscopicus was purified, the N-terminal amino acid sequencing was performed.

[0053] Determine the primers for PCR as follows:

[0054] P1: 5'-GCTAGCGGTGACGACGAGG-3'

[0055] P2: 5'-TTACGGCCAGCCCTGCTTTAC-3'

[0056] Using the above primers, using the extracted total DNA of Streptomyces hygroscopicus CCTCC M203062 as a template, the PCR reaction was carried out in a 50 μL system, and the reaction conditions were denaturation at 95°C for 5 m...

Embodiment 2

[0057] Embodiment 2: Cloning of transglutaminase progenase gene

[0058] The amplified DNA was recovered and purified using Shanghai Sangon UNIQ-10 Column Gel Recovery Kit, 4.5 μL of the purified product and 0.5 μL of the pMD-18T vector were operated according to the kit, connected at 16°C for 12 hours, and the ligated product was heat-shocked Escherichia coli JM109 was transformed. The specific operation is as follows:

[0059] 1) Take the JM109 competent cells out of the -80°C refrigerator and put them on ice quickly, and let them dissolve for 5 minutes.

[0060] 2) 10 μL of the ligation product was added to 100 μL of JM109 competent cells, and placed in ice for 30 minutes.

[0061] 3) After heating at 42°C for 90 seconds, place in ice for 3 minutes.

[0062] 4) Add 950 μL of SOC (preheated at 37° C.) culture medium, and culture with shaking at 37° C. for 90 minutes.

[0063] 5) In the presence of 0.04mg / mL X-Gal (5-bromo-4-chloro-3-indole-α-D-galactoside), 0.024mg / mL IP...

Embodiment 3

[0065] Embodiment 3: Expression of Streptomyces hygroscopicus transglutaminase

[0066] Use restriction endonucleases NcoI and BamHI to cut off from pMD / pro-MTG, and then cut the expression vector pET20b in the same way, then use T4 DNA ligase to carry out ligation reaction, and transform the ligated product into Escherichia coli JM109 first, After obtaining the correct expression plasmid pET / pro-MTG, the expression plasmid was transformed into BL21(DE3) or Rosetta(DE3)pLysS. The engineered bacteria containing the expression plasmid pET / pro-MTG were named BL21 / pET-MTG or Rosetta / pET-MTG.

[0067] To induce expression proceed as follows:

[0068](1) Quickly add 0.1ml of BL21 / pET-MTG or Rosetta / pET-MTG bacterial solution preserved in glycerol tube into a 50ml Erlenmeyer flask containing 25ml of LB medium containing 0.1mg / mL Amp, and cultivate the seeds at 200rpm overnight ( 12h).

[0069] (2) The seeds were inoculated into three 250ml Erlenmeyer flasks containing 50ml of LB m...

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Abstract

The invention relates to a transglutaminase zymogen gene of Streptomyces hygroscopicus and the expression thereof, pertaining to molecular biology field. The invention provides the gene sequence of transglutaminase zymogen of Streptomyces hygroscopicus CCTCC M203062 (China patent: 03152956.9, 2003), and further provides a vector system and a host system that has soluble express of the zymogen of the gene code, a part of soluble expressed zymogen can be obtained through the expression system, and the zymogen can be treated by trypsin to prepare activated transglutaminase.

Description

technical field [0001] The invention relates to a transglutaminase progenase gene of Streptomyces hygroscopicus and its expression, belonging to the field of molecular biology. A novel DNA molecule encoding a transglutaminase proenzyme and its encoded transglutaminase proenzyme are involved. Background technique [0002] Transglutaminase (protein-glutamic acid-γ-glutaminase, Transglutaminase, MTG for short, EC 2.3.2.13), also known as transglutaminase or glutaminyl-peptide γ-glutaminase Amidoacyltransferase can catalyze the intramolecular and intermolecular crosslinking of proteins, the connection between proteins and amino acids, and the hydrolysis of glutamine groups in protein molecules, so as to further improve the functional properties of proteins, increase the nutritional value of proteins, and produce A new type of protein food that meets people's needs, and is known as "21st Century Super Glue". [0003] The application of transglutaminase in food processing mainly...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/78C12N1/20C12R1/55
Inventor 陈坚吴敬堵国成任增亮
Owner JIANGNAN UNIV
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