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Recombinant strains expressing chondroitin 4-sulfotransferase gene and application of recombinant strain

A technology of sulfotransferase and recombinant bacteria, which is applied in the field of bioengineering, can solve the problems of high cost, unfavorable industrial production, and the inability to meet the needs of CSA production, and achieve the effect of high-efficiency expression

Active Publication Date: 2019-06-18
JIANGNAN UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chondroitin can be catalyzed by chondroitin 4-sulfate transferase (C4ST) to synthesize chondroitin sulfate A in the next step. Currently, chondroitin 4-sulfate transferase is mainly extracted from animal cells, and the high cost is not conducive to industrial production
He Wenqin et al. have successfully linked the C4ST derived from H. sapiens through pJK912 plasmid, and performed heterologous expression in P. pastoris host (HeWenqin, Applied Microbiology Biotechnology, 2017, 101:6919-6928; Zhou Zhengxiong, Biotechnology and Bioengineering ,2018,115:1561-1570), but the enzyme activity of C4ST obtained by it is only 6.1U / L, which cannot meet the demand of industrial production of CSA

Method used

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  • Recombinant strains expressing chondroitin 4-sulfotransferase gene and application of recombinant strain
  • Recombinant strains expressing chondroitin 4-sulfotransferase gene and application of recombinant strain
  • Recombinant strains expressing chondroitin 4-sulfotransferase gene and application of recombinant strain

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Experimental program
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Embodiment 1

[0035] Embodiment 1: Construction of recombinant expression vector

[0036] (1) Obtaining the fusion protein MBP-C4ST gene fragment

[0037] In order to enable the high-efficiency expression of the C4ST gene derived from animal cells in microbial cells, it is necessary to fuse maltose binding protein (MBP) from Escherichia coli to the N-terminus of the enzyme.

[0038] Using the artificially synthesized nucleotide sequence such as the MBP gene shown in SEQ ID NO.3 as a template, the MBP gene was amplified using primers MBP-S and RH-MBP-C4ST-A, and the artificially synthesized nucleotide sequence The C4ST gene shown in SEQ ID NO.4 is used as a template, and the C4ST gene is obtained by amplifying the primers RH-MBP-C4ST-S and C4ST-A, and then the fusion PCR technology is used to fuse the MBP gene and the C4ST gene, and finally the primers MBP-S and C4ST-A were amplified to obtain the fusion protein MBP-C4ST gene.

[0039] (2) Construction of pHT101-MBP-C4ST

[0040]Using the...

Embodiment 2

[0050] Embodiment 2: the construction of engineering bacterial strain

[0051] (1) Construction of recombinant Bacillus subtilis

[0052] A single colony of B. subtilis 168 was picked and inoculated into a test tube containing 5 mL of GMI solution, and cultured on a shaker at 200 rpm at 30°C for 16 hours to obtain the first seed solution. Take 2 mL of the first seed solution and transfer it to 18 mL of fresh GMI solution, and incubate at 37° C. for 3.5 hours at 200 rpm to obtain the second seed solution. Transfer 10mL of the second seed solution to 90mL GM II solution, incubate at 37°C and 200rpm for 90min, then centrifuge at 5,000g for 10min at 4°C to collect the bacteria, remove the supernatant but keep 10mL for suspension The bacteria, the suspended bacteria are competent cells and can be directly used for heat shock transformation (45°C, 90S). Add an appropriate amount of recombinant plasmid pHT101-MBP-C4ST to B. subtilis 168 competent cells, culture at 200 rpm at 37°C f...

Embodiment 3

[0055] Example 3: Construction of 3'-phosphoadenosine-5'-phosphorylsulfate PAPS regeneration system

[0056] In the C4ST reaction system, the donor 3'-phosphoadenosine-5'-phosphorylsulfate PAPS of the sulfate group is needed. Therefore, according to the literature (Yuh-Shyong Yang, Protein Expression and Purification, 1996, 8:423-429 ) to express the acylsulfatyltransferase ASTIV, and to construct a 3'-phosphate adenosine-5'-phosphorylsulfate PAPS regeneration system. The specific operation steps are given below:

[0057] (1) Expression of acylsulfatyltransferase ASTIV

[0058] The acylsulfatase transferase gene ASTIV used in the present invention is derived from animal mice. The plasmid pTrcHisA was used as the expression vector, and the artificially synthesized gene fragment ASTIV whose nucleotide sequence was shown in SEQ ID NO.5 was used as the target fragment, and the expression vector pTrcHisA-ASTIV was constructed by enzyme-cut ligation. The expression vector pTrcHis...

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Abstract

The invention discloses recombinant strains expressing a chondroitin 4-sulfotransferase gene and an applicationof the recombinant strain, and relates to the technical field of bioengineering. Chondroitin 4-sulfotransferase gene with the amino acid sequence shown in SEQ ID NO.1 is expressed with Escherichia coli or bacillus subtilis as a host, four recombinant strains are constituted, the high-efficiency expression of chondroitin 4-sulforansferase gene is realized, and final enzyme activity respectively reaches 41.3 U / mL, 32.8 U / mL, 36.5 U / mL and 50.2 U / mL. By using the enzyme as a catalyst, chondroitin sulfate A is catalyzed and synthesis in one step with chondroitin as a substrate, the final conversion efficiency reaches 80%. The method has potential and quite wide values in industrial synthesis of chondroitin sulfate A.

Description

technical field [0001] The invention relates to a recombinant bacterium expressing chondroitin 4-sulfate transferase gene and application thereof, belonging to the technical field of bioengineering. Background technique [0002] Chondroitin sulfate (CS) is a kind of glucuronic acid (D-GlcUA) and N-acetylgalactosamine (GalNAc) linked alternately by β-1,3 bonds and β-1,4 bonds. Straight-chain acidic mucopolysaccharides have various forms of sulfated structures, and the current medicinal ones are mainly A and C isomers. Chondroitin sulfate A (CSA) has a variety of biological activities and medicinal value. It is clinically used to treat rheumatism and arthritis. It can endow cartilage with gel-like properties and anti-deformation ability. It enjoys the title of "soft gold" in the human body. At the same time, CSA can be used as a dietary supplement and moisturizing agent, and is widely used in the fields of food and cosmetics. [0003] At present, the main industrial producti...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/75C12P19/26C12R1/19C12R1/125
Inventor 刘立明张权唐文秀罗秋玲刘佳陈修来
Owner JIANGNAN UNIV
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