Studies On The Expression Of BmSPR Gene In Insect Cells Using The Bac-to-Bac System

Sepiapterin reductase (SPR) is a key enzyme to synthesize tetrahydrobiopterin (BH4) from GTP at the last step. Studies on SPR are mainly in higher vertebrates but rarely in insects. We have previously cloned the gene of SPR in the silkworm Bombyx mori (BmSpr) and made it known that BmSpr is responsible for the phenotype of lemom (lem, a yellow body color larval mutant) and de novo biosynthesis of BH4 is similar between mammals and B. mori. Sepiapterin (SP) is one of endogenous pigments to form the body color of insects, which is contained in the integument of lem larvae in high concentration. BH4 can also be synthesized from SP by the catalyses of SPR and dihydrobiopterin reductase (DHFR), called salvage synthesis pathway. BH4 is an essential cofactor for aromatic acid hydroxylases and nitric oxide synthetase, deficiency of which has been associated with many neuropsychological disorders, hypertension, diabetes, atherosclerosis and other diseases. Recently, SP extraction and purification from lem silkworms, prokaryotic expression of BmSPR and its enzymatic characters have been researched in our lab. We tried to set up an experimental system to synthesize BH4 in vitro by co-expressing BmSPR and BmDHFR.In this study, in order to express BmSpr in insect cells using the Bac-to-Bac/ baculovirus expression system, we have constructed a recombinant plasmid pFastBacHT B-BmSpr and transformed it into DH10Bac competent cells containing different baculovirus particles of AcMNPV or BmNPV. After completing the homologous recombination, two recombinant Bacmids, pAc-BmSpr and pBm-BmSpr were harvested. They were transfected into the insect cells of Sf9 and BmN, respectively, using the transfection kit of Lipofectamine 2000. By multiple infecting, high titer of virus was obtained which was used to infect a large number of insect cells to express recombinant proteins. As a result, SDS-PAGE and western bloting analyses showed that BmSPR were successfully expressed in the cells of both Sf9 and BmN.Bac-to-Bac expression system is effective and safe. Our achievements in this research give experimental basis for futher expressing and purifying recombinant BmSPR using thesystem and help to apply the expressed BmSPR to synthesize BH4 in vitro by using extracted SP from the lem mutant as substrate in the future.