title: An efficient approach for recombinant expression and purification of the viral capsid protein from beak and feather disease virus (BFDV) in Escherichia coli.
abstract: Structural insights into the biology of viruses such as beak and feather disease virus (BFDV) which do not replicate in cell cultures are increasingly reliant on recombinant methods for protein production and purification. Development of efficient methods for homogenous production of BFDV capsid protein is also essential for vaccine development and diagnostic purposes. In this study, two different plasmids (pMCSG21 and pMCSG24), three homologous BFDV capsid proteins, and two unique expression media (auto-induction and IPTG-induced expression) were trialled for over-expression of the BFDV in Escherichia coli. Over-expression was observed for all three recombinant targets of BFDV capsid protein using E. coli BL21 (DE3) Rosetta 2 cell lines under IPTG induction. These proteins could be purified using an optimized, two-step purification process using a buffer containing 20mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 500 mM NaCl and supplemented with 200 mM L-arginine at pH 10.5, to yield a soluble and stable protein of greater than 95% purity. The final concentration of purified protein was approximately fourteen-to-eighteen fold greater than that reported previously. Initial crystallization and X-ray diffraction confirm that the protein is structured in a manner consistent with icosahedral symmetry. Antigenicity of recombinant Cap was confirmed by immunoassay, verifying its validity for use in continued experimentation as a potential DNA vaccine, a reagent in diagnostic assays, and purified concentrated protein for structural and functional biology.
authors.affiliation: School of Animal and Veterinary Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678, Australia; Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Boorooma Street, Wagga Wagga, New South Wales 2678, Australia. Electronic address: ssarker@csu.edu.au.
authors.forename: Subir
authors.initials: S
authors.lastname: Sarker
authors.affiliation: School of Animal and Veterinary Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678, Australia; Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Boorooma Street, Wagga Wagga, New South Wales 2678, Australia. Electronic address: aghorashi@csu.edu.au.
authors.forename: Seyed A
authors.initials: SA
authors.lastname: Ghorashi
authors.affiliation: School of Biomedical Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678, Australia. Electronic address: cswarbrick@csu.edu.au.
authors.forename: Crystall M D
authors.initials: CM
authors.lastname: Swarbrick
authors.affiliation: School of Biomedical Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678, Australia. Electronic address: ykhandokar@csu.edu.au.
authors.forename: Yogesh B
authors.initials: YB
authors.lastname: Khandokar
authors.affiliation: School of Biomedical Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678, Australia. Electronic address: zainab.himiari@gmail.com.
authors.forename: Zainab
authors.initials: Z
authors.lastname: Himiari
authors.affiliation: School of Biomedical Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678, Australia; Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Boorooma Street, Wagga Wagga, New South Wales 2678, Australia. Electronic address: jforwood@csu.edu.au.
authors.forename: Jade K
authors.initials: JK
authors.lastname: Forwood
authors.affiliation: School of Animal and Veterinary Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678, Australia; Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Boorooma Street, Wagga Wagga, New South Wales 2678, Australia. Electronic address: shraidal@csu.edu.au.
authors.forename: Shane R
authors.initials: SR
authors.lastname: Raidal
mesh_terms: D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017925:Circovirus / Q000235:genetics*; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D036022:Capsid Proteins; D003470:Culture Media; D011994:Recombinant Proteins
keywords: Antigenicity; BFDV; Crystallization; IPTG-induction; Protein over-expression; Viral capsid protein