Method for catalytic synthesis of cyclic di-guanosine monophosphate (c-di-gmp) through guanylate cyclase

Abstract

The invention provides a method for catalytic synthesis of cyclic di-guanosine monophosphate (c-di-gmp) through guanylate cyclase. The method specifically comprises the following steps: (1) obtainingof a gene sequence; (2) constructing of a mutation recombinant vector; (3) digestion; (4) conversion; (5) induction expression; (6) degerming for protein collecting; (7) protein purification; (8) protein dialysis; (9) measuring of the protein concentration through a BCA method; and (10) protein in-vitro catalytic reaction. Compared with the prior art, coding protein of a new gene-PA0847 is utilized as c-di-gmp catalytic enzyme; the wild-type PA0847 protein is subjected to point mutation and gene engineering modification; the catalytic enzyme is sufficient in source, and a purification method is stable, simple and convenient; the synthesis process is simple and convenient, and pollution is avoided; and the product yield is stable, and purification is easy.

Description

technical field [0001] The invention relates to a method for catalyzing the synthesis of cyclic guanosine diphosphate by guanylate cyclase. Background technique [0002] According to research, c-di-GMP widely exists in bacteria and participates in the regulation of various complex cellular activities (1), such as biofilm formation (2), virulence (3), motility (4-5), cell differentiation ( 6) and immune regulation during infection (7), etc. [0003] In recent years, researchers have conducted in-depth studies on the immune regulation of its infection process. In 2005, EricB et al. (8) found that c-di-GMP has antibacterial and anti-pathogenic activity, and the therapeutic effect is dose-dependent, indicating that c-di-GMP has considerable potential to develop into a new type of drug, which can be used alone Or used in combination with other medicines to control infection. In 2007, KaraolisDKR et al. (9) treated mouse and human immature reticulocytes with exogenous c-di-GMP ...

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Problems solved by technology

[0013] Chinese patent CN104152472A, titled a kind of diguanylate cyclase gene, carrier, engineering bacteria and its application, discloses that the catalytic enzyme used in the enzymatic method can be obtained from cells; however, when cells are used as enzyme sources to prepare c-di- GMP, the reaction will depend on the enzyme produced by the cell itself; and the active guanylate cyclase (DGC) may be toxic to E. coli, so the controllability of the reaction is poor, and sometimes it is difficult to obtain the exact content of c-di- GMP

[0014] Another obstacle faced by the enzymatic synthesis of c-di-gmp is that after the product binds to certain sites of the enzyme, the guanylate cyclase cannot continue to function, which directly leads to product inhibition and reduced yield (25), To solve this problem, Jenal et al. carried out point mutations on the active PleD protein (26), which requires high experimental techniques

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