Cloning And Expression Of Tetrahydrobiopterin Synthesis-related Enzyme Genes From The Silkworm Bombyx Mori And Analysis Of Enzymatic Properties

It is well established that tetrahydrobiopterin (BH4) is the natural cofactor required forthe hydroxylation of the aromatic amino acids and nitric oxide synthesis, which plays animportant role in neuropsychiatric and endothelial functions. BH4deficiency has beenassociated with many neuropsychological disorders. Many animal experiments and clinicalresearches suggested that BH4deficiency-dependent diseases could be improved byoral addministration of BH4. Because of the chemical synthesis of BH4is difficult, theprice of BH4is very expensive. Sepiapterin (SP)，is one of endogenous pigments to forminsect body color and is contained in the integument of silkworm Bombyx mori mutantlemon (lem) in high concentration. SP is the precursor of BH4in the salvage pathway. Inorder to develop a lower cost, time saving, and higher output pathway than the chemicalsynthesis method to produce BH4in vitro, we cloned and expressed two BH4synthesis-related enzyme genes, SP reductase (SPR) and dihydrofolate reductase (DHFR),from B. mori using prokaryotic expression system and identified the enzymatic propertiesin this research.We firstly transformed the recombinant plasmid pET-24b/BmSPR into E. coli Rosetta(DE3) strain and induced the expression of BmSPR by different concentrations of IPTG atdifferent time points to optimize the protein expression conditions. SDS-PAGE andwestern blot analyses showed that BmSPR could be expressed steadily in prokaryoticexpression system. Then, according to the sequence information from public B. morigenomic and EST databases, we designed specific primers and cloned the silkwormBmDhfr cDNA sequence, ORF of which encodes a185-aa polypeptide with a predictedmolecular mass of about21.1kDa. The ORF was ligated to pET-24b vector for prokaryoticexpression by IPTG induction. Western blot analysis proved that we succeeded inobtaining the recombinant BmDHFR protein.Ni–NTA affinity chromatography was used to purify the recombinant BmSPR andBmDHFR proteins. We optimized the conditions of enzyme reaction by changing thesubstrate concentration, reaction time, temperature, pH of buffer and other factors to assaythe enzymatic activity of the two proteins. The results showed that both BmSPR andBmDHFR exhibited higher enzymatic activities to their substrates SP and dihydrofolate,respectively.Silkworm is one of the most important economic insects, which increasingly becomes an important object for genetic engineering research. In this study, using low-cost E. coliexpression system which could be easily operated and rapidly produce target protein inlarge scale, we obtained recombinant BmSPR and BmDHFR with high purity and goodcatalytic activities. Our findings are of great significance to bio-pharmaceuticallysynthesize BH4in vitro using SP extracted from the lem silkworm mutant as substrate inthe future.