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Method for preparing glycosyl transferase DnMS

A technology of glycosyltransferase, encoding gene, applied in microorganism-based methods, transferases, botanical equipment and methods, etc., can solve the problems of non-expression and insolubility of DnmS

Pending Publication Date: 2022-06-24
GUANGXI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In order to overcome the problem of non-expression and insolubility of glycosyltransferase DnmS, the object of the present invention is to obtain soluble and highly expressed DnmS protein

Method used

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  • Method for preparing glycosyl transferase DnMS
  • Method for preparing glycosyl transferase DnMS
  • Method for preparing glycosyl transferase DnMS

Examples

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Embodiment Construction

[0025] The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention.

[0026] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0027] The vector pET32a and Escherichia coli BL21 (DE3) were collected in our laboratory; the nickel column (Ni NTA Beads6FF) was produced by Tiandiren and the gel chromatography column (Superdex 75prep grade) was produced by cytiva.

[0028] TB liquid medium: 12g tryptone, 24g yeast extract, 4mL glycerol and 0.89M potassium phosphate buffer were dissolved in 1L deionized water. 121℃, high temperature sterilization for 20min.

[0029] Lysis buffer was 50 mM Tris-HCl 300 mM NaCl 10% glycerol, pH 7.4.

[0030] The wash buffer was 50 mM imidazole; the elution buffer was 300 mM imidazole.

[0031] 1. Construction of reco...

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Abstract

The invention discloses a method for cloning, expressing and purifying glycosyl transferase Dnnm. According to the invention, the dnnm gene of the glycosyl transferase is obtained from Streptomyces peuceius ATCC 27952, and a method for cloning, expressing and purifying the glycosyl transferase is provided, and finally, the high-purity glycosyl transferase is obtained under an in-vitro condition for the first time. After the method is implemented, conditions are provided for synthesizing various glycoside products with anticancer activity in an in-vitro enzyme catalysis mode, and a foundation is laid for subsequent glycosyl transferase structure analysis.

Description

technical field [0001] The invention belongs to the technical field of antibiotic medicine, in particular to a method for preparing glycosyltransferase DnmS. Background technique [0002] The antitumor drug doxorubicin is a representative member of anthracyclines, consisting of a conserved tetracyclic aglycone, 7,8,9,10-tetrahydro-5,12-naphthoquinone and various TDP-deoxysugars composition. The sugar moiety of the molecule is assembled by specialized glycosyltransferases (Gtfs), which greatly affects the biological activity and product diversity of the glycoside product. However, biochemical studies of natural product Gtfs are hindered by the difficulty in obtaining high-purity active Gtfs, aglycones, and deoxysugars. In the early stage of this experiment, a new E. coli fusion plasmid pGroE was constructed to help the soluble expression and purification of the glycosyltransferase DnmS in the biosynthetic pathway of Streptomyces peucetius ATCC 27952, and finally a high-puri...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12R1/19
CPCC12N9/1048C12N15/70
Inventor 潘丽霞周会敏颜以金梁智群陈桂光曾伟
Owner GUANGXI UNIV
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