title: Codon optimization enhances protein expression of Bombyx mori nucleopolyhedrovirus DNA polymerase in E. coli.
abstract: Bombyx mori nucleopolyhedrovirus (BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms, while BmNPV DNA polymerase (BmNPV-pol), encoded by ORF53 gene, plays a central role in viral DNA replication. Efficacy studies of BmNPV-POL are limited because of poor heterologous protein expression in E. coli. Here, we redesigned the BmNPV-pol to preferentially match codon frequencies of E. coli without altering the amino acid sequence. Following de novo synthesis, codon-optimized BmNPV-pol (co-BmNPV-pol) gene was cloned into pET32a and pGEX-4T-2 vector. The expression of co-BmNPV-POL in E. coli was significantly increased when BmNPV-POL was fused with GST protein rather than a His-tag. The co-BmNPV-POL fusion proteins were isolated using GST affinity chromatography and Mono Q iron exchange chromatography. Protein purity and identity were confirmed by western blot and MALDI-TOF analyses. The biological activity of purified proteins was measured on a poly(dA)/oligo(dT) primer/template. The specific polymerasing activity of the recombinant BmNPV-POL was 6,329 units/mg at optimal conditions. Thus, a large amount of purified protein as a soluble form with high activity would provide many benefits for the functional research and application of BmNPV-POL.
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang, 212013, Jiangsu, People's Republic of China.
authors.forename: Huifang
authors.initials: H
authors.lastname: Song
authors.forename: Guohui
authors.initials: G
authors.lastname: Li
authors.forename: Weijun
authors.initials: W
authors.lastname: Mai
authors.forename: Guoping
authors.initials: G
authors.lastname: Huang
authors.forename: Keping
authors.initials: K
authors.lastname: Chen
authors.forename: Yajing
authors.initials: Y
authors.lastname: Zhou
authors.forename: Huiqing
authors.initials: H
authors.lastname: Chen
mesh_terms: D000818:Animals; D012831:Bombyx / Q000821:virology; D003062:Codon; D004247:DNA / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D017924:Nucleopolyhedroviruses / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D014764:Viral Proteins; D004247:DNA; D004259:DNA-Directed DNA Polymerase