title: Effect of tandem rare codon substitution and vector-host combinations on the expression of the EBV gp110 C-terminal domain in Escherichia coli.
abstract: Gp110 of Epstein-Barr virus (EBV) is a glycoprotein that functions exclusively during the assembly of EBV nucleocapsid and the release of infectious EBV. Its C-terminal tail domain (gp110 CTD) is essential for gp110's function and may provide signals that are responsible for the assembly and release of EBV. In the present study, to get large amounts of gp110 CTD for structural analysis, the effects of vector system, codon usage, and host strain on expression levels of gp110 CTD in Escherichia coli have been investigated. The coding region of gp110 CTD (11 kDa) was subcloned into the expression vectors pSE 280, pET-15b, pET-29a, pMAL-c2x, and pGEX-4T-1. Except the pMAL-c2x construct, all the others failed to express detectable amounts of recombinant gp110 CTD. Substituting a tandem rare AGA (Arg) codon with a synonymous CGC (Arg) codon facilitated expression of the recombinant protein, while a protease-deficient host E. coli strain helped in the accumulation of a soluble form of gp110 CTD fusion. The secondary structures of the obtained recombinant gp110 CTD purified from soluble extracts and inclusion bodies were compared using circular dichroism analysis. In aqueous solutions, both samples equally adopt a mixed alpha-helix and beta-sheet conformation as well as a partly unordered structure. Notably, in the membrane-mimicking environments the helical propensity of gp110 CTD increased up to the previously predicted level based on its sequence, suggesting that gp110 CTD may fold into a more stable conformation through interactions with the cell membrane.
authors.affiliation: Research Institute of Pharmaceutical Sciences, College of Pharmacy, San 56-1, Shillim-Dong, Kwanak-Gu, Seoul 151-742, Korea.
authors.forename: Sung Jean
authors.initials: SJ
authors.lastname: Park
authors.forename: Suk Kyeong
authors.initials: SK
authors.lastname: Lee
authors.forename: Bong Jin
authors.initials: BJ
authors.lastname: Lee
mesh_terms: D002942:Circular Dichroism; D004926:Escherichia coli; D005822:Genetic Vectors; D015151:Immunoblotting; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification
chemical_list: C060207:Epstein-Barr virus glycoprotein gp110; D014764:Viral Proteins