The Construction And Expression Of Denitrosylation Enzymes Cloning And Expression Of Genes Of Inonotus Obliquus

The medicinal basidiomycete Inonotus obliquus produces medically significant polyphenols yet grows very slowly in its natural habitats. Under laboratory culture, however, this fungus is able to accumulate quickly large sums of mycelial biomass but little polyphenols. It has been known that under lab condition, the enzyme involved in biosynthesis of polyphenols are S-nitrosylated by nitric oxide(NO) and thereby their activities are reduced. Thioredoxin(Trx) and thioredoxin reductase(TrxR) can increase the polyphenol accumulation by denitrosylation only when nitroso glutathione reductase(GSNOR) was inhibited. However, the molecular mechanism of Trx/ TrxR denitrosylation and mechanism of GSNOR constraints Trx / TrxR denitrosylation and mechanism of GSNOR constrains Trx/TrxR denitrosylation remains to be determined.Based on the sequence of Inonotus obliquus, we designed specific primers of the genes encoding Trx, TrxR and GSNOR, and constructed plasmids using expression vector PGEX-4T-3 with GST tag. The constructed recombinant expression plasmids PGEX-Trx, PGEX-TrxR and PGEX-GSNOR were transformed into E. coli BL21, and then induced by IPTG, purified with Glutathione S-transferase affinity chromatography. This study established a method for testing the molecular mechanism of Trx/TrxR denitrosylation and mechanism about GSNOR constraints Trx/TrxR denitrosylation. In addition, this study also established a vitro model to verify the catalytic activity of the above three fusion proteins, in the process of denitrosylation. In this model, the recombinant plasmid PGEX-STS/pGEX-4CL1 was constructed by one step cloning technology, and the fusion protein GST-STS/GST-4CL1 were produced by E. coli in the presence of IPTG induction. The results provide insights into the elucidation of interaction mechanisms for S-denitrosylation.