Expression and single-step purification of enzymatically active vaccinia virus thymidine kinase containing an engineered oligohistidine domain by immobilized metal affinity chromatography.
A method has been developed for the controlled expression in Escherichia coli and rapid purification of an enzymatically active vaccinia virus (VV) thymidine kinase protein containing an engineered oligohistidine domain. The nucleotide sequence that encodes the VV thymidine kinase open reading frame was inserted into a plasmid expression vector (pET-16b, Novagen Inc., Madison, WI) under the control of a strongly repressed bacteriophage T7 promoter and high efficiency translational signals. The construct (pET-16b:TK) directs the synthesis of a fusion protein (His-TK) with an N-terminal histidine decapeptide fused to the VV thymidine kinase polypeptide. Upon induction of E. coli strain BL21(DE3)pLysS with isopropyl beta-D-thiogalactoside, accumulation of large quantities of a 22-kDa protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein reacted with polyclonal antiserum raised against a TrpE-VVTK fusion protein. The predominantly soluble fusion protein (approximately 13% of the total soluble bacterial protein) was purified to homogeneity from crude bacterial extracts in a single-step by immobilized metal chelate affinity chromatography (Ni(2+)-nitrilotriacetic acid-agarose) under nondenaturing conditions and was shown to have thymidine kinase activity. The yield of the purification scheme was about 15 mg recombinant protein/liter of bacterial culture. The availability of purified VV TK protein should greatly facilitate biochemical studies on its enzymatic activity, as well as analyses of its structural and functional domains.
Franke
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Department of Microbiology, Oregon State University, Corvallis 97331-3804.
Hruby
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D000595:Amino Acid Sequence; D002474:Cell-Free System; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics
D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D013937:Thymidine Kinase