Cloning, expression, and purification of histidine-tagged preS domains of hepatitis B virus.
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 6x His tag at the carboxy-terminal end of the polypeptides. Codons for the 6x 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-beta-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 6x 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.
Núñez
E
E
Departamento de Bioquímica y Biología Molecular, Universidad Complutense, 28040 Madrid, Spain.
Wei
X
X
Delgado
C
C
Rodríguez-Crespo
I
I
Yélamos
B
B
Gómez-Gutiérrez
J
J
Peterson
D L
DL
Gavilanes
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006514:Hepatitis B Surface Antigens / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D006515:Hepatitis B virus / Q000235:genetics*; D006639:Histidine; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012709:Serum Albumin / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; D011994:Recombinant Proteins; D012709:Serum Albumin; C050806:presurface protein 1, hepatitis B surface antigen; C050807:presurface protein 2, hepatitis B surface antigen; D006639:Histidine