Cloning, expression, and purification of histidine-tagged preS domains of hepatitis B virus

Citation
E. Nunez et al., Cloning, expression, and purification of histidine-tagged preS domains of hepatitis B virus, PROT EX PUR, 21(1), 2001, pp. 183-191
Citations number
37
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEIN EXPRESSION AND PURIFICATION
ISSN journal
1046-5928 → ACNP
Volume
21
Issue
1
Year of publication
2001
Pages
183 - 191
Database
ISI
SICI code
1046-5928(200102)21:1<183:CEAPOH>2.0.ZU;2-2
Abstract
The preS domains of the hepatitis B virus are hydrophilic polypeptides that have been implicated, among other functions, in the binding of the virus t o hepatocytes and in the induction of virus-neutralizing antibodies. A meth od of overproducing the preS domains of two different subtypes, adw and ayw , has been developed by adding a 6 x His tag at the carboxy-terminal end of the polypeptides. Codons for the 6 x His were added in reverse primers use d to amplify the corresponding cDNAs, The polymerase chain reaction product s were cloned into the expression vectors pET-3d (subtype ayw) and pT7-7 (s ubtype adw), under the control of the inducible bacteriophage T7 RNA polyme rase promoter. Upon induction with isopropyl-beta -D-thiogalactopyranoside, proteins were overexpressed and purified by affinity chromatography on a N i-nitrilotriacetic acid agarose column. This method yielded 20-40 mg of hig hly pure and very stable proteins per liter of cell culture. Circular dichr oism and fluorescence spectroscopy of isolated preS-his-ayw and preS-his-ad w, as well as their ability to bind polymerized human serum albumin, indica te that the 6 x His tag does not modify the native-like conformation and, t herefore, they may be considered as useful tools to study the function of t hese viral polypeptide regions. (C)2001 Academic Press.