Cloning And Expression Of The Truncated Human Hepatic Squalene Synthase And High Density Fermentation Of The Recombinant Squalene Synthase In E.coli

Hypercholesterolaemia is a major risk factor for the generation and development of atherosclerosis and coronary heart disease(CHD).In 1984,it was demonstrated for the first time that there exists a link between serum cholesterol levels and risk to CHD.The inhibition of cholesterol biosynthesis constitutes an important approach to the reduction of serum cholesterol levels.The 'statins',a family ofHMG-CoA reductase(HMGR)inhibitors,are the most common cholesterol-lowering drugs.They reduce cardiovascular disease morbidity and mortality with a high level of safety.Nonetheless,Statins suppress all post-mevalonate biosynthetic pathways,not noly cholesterol but also other important nonsteroidal isoprenoids(e.g.dolichol,ubiquinone,isopentenyl tRNA and prenylated proteins), inevitablely.Selective inhibition of cholesterol biosynthesis is a desirable pharmaceutical goal.Squalene Synthase(farnesyl-diphosphate farnesyltransferase,SQS, EC2.5.1.21)is unique to cholesterol biogenesis,and inhibition would lower serum cholesterol levels but avoid any potential adverse effect associated with reduced synthesis of the nonsteroidal isoprenoids by the inhibitors of HMGR.Therefore SQS has been potential targets for the design of cholesterol-lowering drugs. In this article,a 1020 bp trhncated squalene synthase(tSQS)cDNA was cloned from HepG2 cells by reverse transcription-polymerase chain reaction(RT-PCR).The truncated cDNA was subcloned into prokaryotic expression vector pET28a(+)and the constructed plasmid was introduced to Escherichia coli strain BL21(DE3)for induced overexpression.Then the recombinant tSQS was purified by Ni²⁺affinity chromatograph.Activity of recombinant tSQS was determined by mensurated absorbtion value of NADPH at double wavelengths(340/384 nm).The high cell density cultivation techniques of E.coli BL21(pET28a- tFDFT1)was studied.The effects of the composition of the fermentation medium,induction time,and carbon sources on the expression level of recombinant tSQS and cell output were analyzed.When cultured in modified M9 medium at 25℃with glycerol as the carbon sources,the recombinant E.coli expressed recombinant tSQS at the level up to 30.24%of the total proteins.The active recombinant tSQS(41KDa)was obtained,and the method for determining squalene synthase activity was established.These results will establish the foundation of abundant preparation of recombinant SQS,establishment of SQS inhibitors high-throughput drug screening model and discovery of hypocholesterolemic drugs targeting SQS.