Expression, Purification And Functional Study Of PNGase F And Preparation Of Polyclonal Antibody Of Sodium Dependent Neutral Amino Acid Transporter1(SNAT1)

N-glycosidase F (PNGase F) is a enzyme of deglycosylation, secreted by thegram-negative bacterium Flavobacterium meningoseptic’um. Its theoreticalmolecular weight is about34.8kDa. The enzyme has a very broad substratespecificity. PNGase F hydrolyzes all types of asparagine-linked oligosaccharides,provided that both the amino and carboxyl groups of the asparagine are inpolypeptide linkage. It recognizes mainly the inner di-N-acetylchitobiose moietyof the carbohydrate chain. In this study, the PNGase F gene of945bp wasampliifed by PCR technique. A prokaryotic expression vector, pET28a-PNGase F,was constructed by digestion and ligation. After E.coli (BL21) containingpET28a-PNGase F plasmids was cultured and induced by IPTG at16°C, solublePNGase F was puriifed through Ni column, and the purity is up to over95%bySDS-PAGE identification. It played a certain role in the future study of PNGase F.Sodium-coupled neutral amino acid transporter belong to the SLC38family.SLC38family is divided into System A and System N. SNAT1and SNAT2belongto System A, SNAT3belonging System N. It has been reported that alterations inglutamate transporters is responsible for several neurodegenerative disorders. Weconstructed pBK-CMVA-SNAT3-HA plasmid by molecular cloning techniquesand proved SNAT3-HA fusion protein expressed and localized on the cellmembrane normally by Western blot technique. Deglycosylation of purifiedPNGase F on neutral amino acid transporter SNAT1，SNAT2and SNAT3showedthat PNGase F is highly effective and SNAT1, SNAT2，SNAT3containglycosylated sites.Hydropathy analysis predicts that SNAT1contains11transmembrane helices(TM) with an intracelluar N terminus and an extracelluar C terminus.We predictedthe potential N-glycosylation sites of SNAT1. After the asparagine of these twosites were mutated into glutamine, we studied the number and location of N-glycosylation sites of SNAT1by Western blot technique. The result of mobilityshift on SDS-PAGE showed that there are twe N-glycosylation sites in SNAT1，N251and N257. It has played an important supporting role for the further researchof SNAT1membrane topology.Currently, there is no effective SNAT1specific antibody, which greatlyhindered the depth study of the structure and function of SNAT1. In this study,pET28a-SNATl-75aa plasmids was constructed by molecular cloning techniques.After expression and purification of SANTl-75aa protein, it was used to immunizeNew Zealand white rabbit in order to obtain SNAT1polyclonal antibody. SpecificSNAT1antibody was obtained after the serum was purified by Thiopropylsepharose6B column. This provides a useful tool for the future depth study ofstructure and function of SNAT1.