Overexpression and purification of the hepatitis B e antigen precursor.
Circumstantial evidence suggests that the secreted hepatitis B virus (HBV) e antigen (HBeAg) and/or its 22 kDa precursor (P22) have an essential role in the establishment of persistent infection. In order to identify cellular proteins that could interact with P22, large amounts of this protein are required to perform pull-down assays. A plasmid was constructed encoding a recombinant P22 with a Histidine-tag at its N-terminal extremity (P22r). The initial attempts to overexpress P22r in a conventional Escherichia coli strain failed, most likely due to the presence of rare AGA/AGG codon clusters in the 3' part of the gene. To overcome this difficulty, P22r was overexpressed in the Epicurian coli BL21-codonplus (DE3)-RIL strain, which possesses extra copies of the ArgU gene that encodes the tRNA(AGA/AGG). In this strain, P22r was overexpressed successfully and then purified in milligram quantities by metal affinity chromatography on Ni2+-chelated His-Bind resin. The purified recombinant protein P22r was able to interact with a cellular protein (P32), which had previously been shown to co-immunoprecipitate with native P22, indicating that at least some of the P22r molecules were folded correctly.
Lainé
Sébastien
S
Laboratoire de Génétique des Virus, UPR 9053-CNRS, Avenue de la Terrasse, 91198, Gif sur Yvette, France
Salhi
Samia
S
Rossignol
Jean-Michel
JM
D001483:Base Sequence; D002846:Chromatography, Affinity; D003062:Codon / Q000235:genetics; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D006513:Hepatitis B e Antigens / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D017510:Protein Folding; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D012329:RNA, Bacterial / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D003062:Codon; D006513:Hepatitis B e Antigens; D011498:Protein Precursors; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins