Study On The Expression Of Cyclooxygenase In Rhodospirillum RubrumH2

Cyclooxygenase(COX)is the central enzyme in the biosynthetic pathway from arachidonic acid to prostaglandin.It has two isoenzymes:COX-1 and COX-2.Both are membrane proteins and are water-soluble.This study mainly explored COX-1 in order to express active COX-1 in the photosynthetic bacteria Rhodospirillum rubrumH2(R.rubrumH2)expression system.In addition,COX-1 and microsomal prostaglandin E synthase-1(mPGES-1)can react with Dohomo-γ-Linolenic Acid(DGLA)to produce prostaglandin E1(Alprostadil E1,PGE1),also known as alprostadil,it is a naturally occurring prostaglandin that can be used as a medicine,so it is tried to express COX-1,which lays the foundation for the synthesis of PGE1.In this study,COX-1 was used as the research object.First,pPUCTerm-COX-1 expression vector was constructed by genetic recombination,and pPUCTerm-mPGES-1 expression vector was constructed on this basis,which provided the basis for subsequent detection of COX-1 activity.Using R.rubrumH2 as a membrane protein expression host,according to the nature of E.coli WM3064 binding transformation,the constructed vector was transformed into R.rubrumH2.SDS-PAGE and electron microscopy were used to detect the expression of COX-1 and mPGES-1.HPLC and LC-MS were used to detect the activity of COX-1.The effects of substrate concentration,reaction time,and reaction pH on the activity of COX-1 during the enzymatic reaction were studied.The results showed that the recombinant strains pPUCTerm-COX-1/mPGES-1 identified by PCR,double digestion,and gene sequencing were successfully constructed;COX-1 and mPGES-1 were identified in photosynthetic bacteria by SDS-PAGE and electron microscopy,successfully expressed in the R.rubrumH2 expression system,their sizes are:53.8 kDa,17 kDa,and both exist as membrane proteins in R.rubrumH2;COX-1 was successfully expressed in R.rubrumH2 by HPLC and LC-MS and has active;COX-1 has the highest activity during the enzymatic reaction when the substrate concentration is 9.674 mM / mL,the reaction time is 30 min,and the reaction pH is 8.0,and the content of PGE1 generated was 0.0482 mg / mL,0.0529 mg / mL,0.046 mg / mL.This study successfully expressed active COX-1 in the Rhodospirillum rubrumH2 expression system,providing a basis for COX-1 for biotransformation and synthesis of PGE1,and strongly promoted the development of membrane protein research.