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Construction method and application of genetically-engineered bacterium for expressing prenyltransferase ComQ

A genetically engineered bacteria and isopentenyl technology, applied in the field of genetically engineered protein engineering, can solve the problems of low content, limited natural sources and distribution, and difficult chemical synthesis.

Inactive Publication Date: 2018-09-04
DALIAN NATIONALITIES UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the natural sources and distribution of this class of compounds are extremely limited, and the content is extremely small
The chemical synthesis of such compounds is also very difficult, involving chemoselectivity, site selectivity, the number of prenylations, etc. during the synthesis process, and the reaction conditions are relatively harsh.

Method used

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  • Construction method and application of genetically-engineered bacterium for expressing prenyltransferase ComQ
  • Construction method and application of genetically-engineered bacterium for expressing prenyltransferase ComQ
  • Construction method and application of genetically-engineered bacterium for expressing prenyltransferase ComQ

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 Construction of genetically engineered bacteria expressing prenyltransferase ComQ

[0034] (1) Design primers for isopentenyltransferase ComQ

[0035] According to the base sequence of the ComQ protein registered by NCBI and the multiple cloning restriction site of the vector pProEX-HTa, the upstream and downstream primers were designed by using the clone manager software, and NcoI and XbaI restriction sites were respectively introduced. The primer information of ComQ is as follows: upstream primer F-ComQ: 5'-GGGCCATGGATATGTGTTTAGATGCCGAA-3' (the underlined part is NcoI restriction site); downstream primer R-ComQ: 5'-GGGTCTAGATTATTTTTCATCCTCCTCTAA-3' (the underlined part is XbaI Restriction sites).

[0036] (2) Amplify the target gene of isopentenyltransferase ComQ

[0037] Using the Staphylococcus aureus genome as a template, add upstream and downstream primers, Taq DNA polymerase and buffer to the system for PCR reaction. The PCR parameters are: pre-denat...

Embodiment 2

[0044] Embodiment 2 utilizes genetically engineered bacteria to express isopentenyl transferase ComQ

[0045] (1) Preparation of target protein crude extract

[0046]The bacteria were inoculated into the liquid LB medium containing ampicillin with a final concentration of 100mg / mL according to the inoculation amount of 1vt%, and cultured with shaking at 37°C. When the OD600 was 1.0, IPTG with a final concentration of 0.5mM / L was added to induce , 30 ° C after 8h culture collected bacteria. The collected bacteria were suspended with buffer (20mM Tris-HCl (pH 8.0), 150mM NaCl), ultrasonically disrupted, centrifuged at 13000rpm for 50min, and the supernatant was saved as a crude extract for future use.

[0047] (2) Preliminary separation and purification of the crude extract of the target protein by metal ion chelation chromatography

[0048] 5mL of the treated filler (Ni Sepharose TM High Performance, GE Healthcare TM ) into a low-pressure chromatography column (Bio-Rad, mo...

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Abstract

The invention relates to a construction method and application of a genetically-engineered bacterium for expressing prenyltransferase ComQ, and belongs to the technical field of gene engineering and protein engineering. The method is mainly characterized by including the steps of designing a primer of the prenyltransferase ComQ, amplifying target genes of the prenyltransferase ComQ, constructing recombinant plasmid through an enzyme digestion and enzyme linking method, identifying the recombinant plasmid and constructing a genetically-engineered bacterial strain through chemical transformation. By means of the method, the genetically-engineered bacterial strain for expressing the prenyltransferase is constructed for biosynthesis of various aromatic compounds with the pharmacological activity and isopentenylated peptides compounds by means of the prenyltransferase, and the genetically-engineered bacteria are used for heavily expressing, separating and purifying the prenyltransferase; bymeans of the prenyltransferase which is obtained through separation and purification, isopentenylated treatment can be conducted on aromatic radicals in peptide compounds so that the method can be used for isopentenylated treatment of the aromatic compounds.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering protein engineering, and specifically relates to a construction method and application of a genetic engineering bacterium expressing isopentenyl transferase ComQ. Background technique [0002] In recent years, prenyl aromatic compounds have attracted more and more attention because of their structural diversity and good pharmacological activity. Many studies have shown that this type of compound has a wide range of pharmacological activities such as anti-oxidation, hypolipidemic, anti-osteoporosis, anti-tumor, anti-virus, and anti-bacterial. Prenyl aromatic compounds have better biological activity, which is derived from the fact that the isopentenyl group substituted on the aromatic compound skeleton increases its lipophilicity, thereby enhancing the affinity between the compound molecule and the biological membrane. However, the natural sources and distribution of this class of comp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N9/10C12R1/19
CPCC12N9/1085C12N15/70C12Y205/01
Inventor 权春善王路路许永斌张丽影郑维周伟陈苛蒙
Owner DALIAN NATIONALITIES UNIVERSITY
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