In silico analysis and experimental study on Bst DNApolymerase ۱ Large fragment (Bst pol ۱ L.F) in E.coli

Background and Aim : Nowadays, isothermal amplification techniques like loop mediatedisothermal amplification (LAMP), are getting more attention in comparison to other common DNAamplification methods same as PCR, due to their benefits. LAMP’s enzyme is a type of DNApolymerase۱ which was extracted from Geobacillus stearothermophilus strains and termed Bst pol۱. This paper is aimed to study in silico analysis in order to clone and to express Bst pol۱ largefragment (Bst pol۱ L.F) in E.coliBL۲۱ (DE۳) plysS.Methods : The nucleotide sequence encoding Bst pol۱ L.F was obtained from NCBI. Secondarystructure consensus prediction was performed using the SPOMA method, PSIPRED, ProtParam,Pepstats, Scratch Protein Prediction, and Recombinant Protein Solubility Prediction. Moreover,two strains of Geobacillus stearothermophilus PTCC۱۷۱۳ and IBRC-M۱۰۷۷۱were obtained frominternal resources, and three pairs of primers were designed including diagnostic primers andcloning primers. They degenerated primers to investigate the Bstpol۱L.F gene in Iranian nativestains.Results : The codon usage bias in E. coli was upgraded the CAI from ۰.۷۱ to ۰.۸۷, and GC contentwas optimized to increase the half-life of the mRNA. A secondary structure study showed Bst pol۱L.F is a hydrophobic protein and its solubility upon overexpression with a probability of ۰.۸۸۲۰۷۴and possibility of expression in inclusion bodies of ۰.۷۹۸. The aliphatic index computed byExPasy’s ProtParam showed that mRNA was efficiently stable for translation in the host. Theencoding sequence was optimized and synthesized, and a pET-۲۸a (+)-Bst pol۱ L.F (pEBpol۱ L.F)recombinant vector was produced. The cloning was confirmed by PCR and double digestiontechniques. The accuracy of expression in transformants was analyzed by applying SDS-PAGEand Western blotting which showed a band around ۶۶.۶۲ kDa associated with Bst pol۱ L.F protein.Conclusion : No gene was detected using designed primers in native strains, indicating that maybethere is some difference between the Bst pol۱ L. F gene sequence in native strains versus the genesubmitted in NCBI data bank

کلیدواژه ها:

cloning and expression ، Bst pol۱ L. F ، Geobacillus stearothermophilus ، Loopmediated isothermal amplification (LAMP) ، in silico