Cloning, Expression, and Purification of Histidine-Tagged preS Domains of Hepatitis B Virus

• Published in: Protein expression and purification 2001-02, Vol.21 (1), p.183-191
• Main Authors: Núñez, Elena, Wei, Xin, Delgado, Carmen, Rodrı́guez-Crespo, Ignacio, Yélamos, Belén, Gómez-Gutiérrez, Julián, Peterson, Darrell L., Gavilanes, Francisco
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Binding Sites; Circular Dichroism; Cloning, Molecular - methods; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Hepatitis B Surface Antigens - chemistry; Hepatitis B Surface Antigens - genetics; Hepatitis B Surface Antigens - isolation & purification; Hepatitis B virus - genetics; Histidine; Humans; Molecular Sequence Data; Polymerase Chain Reaction; Protein Precursors - chemistry; Protein Precursors - genetics; Protein Precursors - isolation & purification; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Sequence Alignment; Sequence Homology, Amino Acid; Serum Albumin - chemistry; Spectrometry, Fluorescence
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.2000.1368

The preS domains of the hepatitis B virus are hydrophilic polypeptides that have been implicated, among other functions, in the binding of the virus to hepatocytes and in the induction of virus-neutralizing antibodies. A method of overproducing the preS domains of two different subtypes, adw and ayw, has been developed by adding a 6× His tag at the carboxy-terminal end of the polypeptides. Codons for the 6× His were added in reverse primers used to amplify the corresponding cDNAs. The polymerase chain reaction products were cloned into the expression vectors pET-3d (subtype ayw) and pT7-7 (subtype adw), under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl-β-d-thiogalactopyranoside, proteins were overexpressed and purified by affinity chromatography on a Ni–nitrilotriacetic acid agarose column. This method yielded 20–40 mg of highly pure and very stable proteins per liter of cell culture. Circular dichroism and fluorescence spectroscopy of isolated preS-his-ayw and preS-his-adw, as well as their ability to bind polymerized human serum albumin, indicate that the 6× His tag does not modify the native-like conformation and, therefore, they may be considered as useful tools to study the function of these viral polypeptide regions.