An expression cassette for recombinant expression of echinocandin b deacylase and its application

A technology of deacylase and echinocandin, applied in the field of bioengineering, can solve the problems of inactive protein, Escherichia coli growth inhibition, unsatisfactory activity and solubility, etc., and achieve the effect of high-efficiency expression

Active Publication Date: 2022-04-29
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its activity and solubility are not ideal
A large number of inclusion bodies accumulated in the periplasmic space, and the inactive protein even hindered the growth of E. coli

Method used

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  • An expression cassette for recombinant expression of echinocandin b deacylase and its application
  • An expression cassette for recombinant expression of echinocandin b deacylase and its application
  • An expression cassette for recombinant expression of echinocandin b deacylase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Construction of recombinant strain E.coli BL21(DE3) / pT7-α / β.

[0031] 1. Codon optimization and synthesis of the gene encoding echinocandin B deacylase

[0032] The echinocandin B deacylase was obtained from a plasmid preserved in the laboratory.

[0033] According to the codon preference of Escherichia coli, the codon-optimized echinocandin B deacylase coding nucleotide (DNA) sequence was artificially designed and synthesized, and the target gene fragment was obtained after sequencing verification.

[0034] The amino acid sequence of echinocandin B deacylase is shown in SEQ ID NO.1, and its optimized nucleic acid sequence is shown in SEQ ID NO.2, wherein the signal peptide coding sequence is 1-96bp, and the alpha subunit coding sequence is 97 -645bp (optimized sequence, the nucleotide sequence after adding TAA as the stop codon is shown in SEQ ID No.6), the connecting peptide coding sequence is 646-690bp, and the β subunit coding sequence is 691-2364bp (after optimizi...

Embodiment 2

[0055] Construction of recombinant strains T7-α / β-6K, T7-GE-α / β-6K, T7-GK-α / β-6K, pT7-GE-α / β.

[0056] 1. Construction of recombinant strains containing different lytic short peptides

[0057] Wherein the nucleic acid sequence of the short peptide is:

[0058] 6K solubilizing short peptide (amino acid sequence KKKKKK): aaaaaaaaaaaaaaaaaaa;

[0059] GE solubilizing short peptide (amino acid sequence GEGEG): ggagaaggagaagga;

[0060] GK solubilizing short peptide (amino acid sequence GKGKG): ggtaaaggtaaaggt.

[0061] The primer sequences used are:

[0062] GKGKG Fusion-F:

[0063] AGCGCAGGCCggtaaaggtaaaggtCATGATGGTGGCTACGCGGCGCTGAT;

[0064] GKGKG Fusion-R:

[0065] GCGCCGCGTAGCCACCATCATGacctttaccttaccGGCCTGCGCTACGGTAGCGAAA;

[0066] GEGEG Fusion-F:

[0067] TAGCGCAGGCCggagaaggagaaggaCATGATGGTGGCTACGCGGCGCTGATCCG;

[0068] GEGEG Fusion-R:

[0069] GCGCCGCGTAGCCACCATCATGtccttctccttctccGGCCTGCGCTACGGTAGCGAA;

[0070] 6K-F:

[0071] GAAGCGCCGGACGCGAAAAAAAAAAAAAAAAAATAAg...

Embodiment 3

[0080] Optimization of fermentation conditions of recombinant strain T7-GE-α / β-6K.

[0081] 1. Optimization of the inducer concentration of the recombinant strain T7-GE-α / β-6K.

[0082] It is well known that, in addition to the expression host itself, expression conditions largely affect the productivity of recombinant enzymes. Therefore, we investigated the effects of IPTG concentration, induction temperature and induction time on the activity of ECBD-expressing crude enzyme solutions. Enzyme activity increased with increasing IPTG concentration from 0 mM to 0.4 mM, but further increases in IPTG concentration decreased the activity. When the concentration of IPTG exceeds 0.6 mM, the soluble forms of α and β subunits are significantly reduced. The viability of the cells correlates with the expression level of the soluble form of the alpha subunit. The strongest growth inhibition occurred at 0.2 mM IPTG. The highest enzyme activity was obtained at 0.4mM IPTG ( Figure 5 ),...

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Abstract

The invention discloses an expression cassette for recombinantly expressing echinocandin B deacylase and its application. The expression cassette includes a promoter nucleic acid sequence connected in sequence, an RBS nucleic acid sequence, a nucleic acid sequence encoding an OmpA signal peptide, an encoding The nucleic acid sequence of the first solubilizing peptide, the nucleic acid sequence encoding the α subunit of echinocandin B deacylase, the first stop codon, the RBS nucleic acid sequence, the nucleic acid sequence encoding the OmpA signal peptide, the encoding echinocandin B deacylase The nucleic acid sequence of the β subunit of the acylase, the nucleic acid sequence encoding the second lytic peptide, and the second stop codon, wherein the amino acid sequence of the first lytic peptide is: GEGEG; the amino acid sequence of the second lytic peptide is: KKKKKK . The expression cassette of the present invention can be used to express and obtain echinocandin B deacylase, through the addition of two specific sequences of prolytic peptides, the echinocandin B deacylase can be effectively and efficiently expressed, and the bacteria obtained by fermentation culture After crushing, the enzyme activity can be as high as 57.88U / g.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an expression box for recombinantly expressing echinocandin B deacylase and its application. Background technique [0002] Fungal infection refers to an infection caused by fungal microorganisms. In recent years, the number of deaths caused by fungal infections has been increasing, which is related to the wide application of corticosteroids, cytotoxic drugs, spectrum antibiotics and immunosuppressants in clinical practice. Echinocandins, as inhibitors of cell wall synthetases, generally have strong antibacterial activity and no antagonism with other drugs. Among them, anidungin is the most prominent, which has a large distribution volume and spectrum Antibacterial properties, no hepatic metabolism, no cross-resistance, etc., and the catalytic production of anidungin precursors by biological methods has become a hot spot in the research of antifungal drugs at home and abro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/62C12N1/21C12N9/80C12R1/19
CPCC12N15/70C12N9/80C12N2800/22C07K2319/02C07K2319/00
Inventor 柳志强邹树平郑裕国牛坤韩鑫朱寒悦
Owner ZHEJIANG UNIV OF TECH
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