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Title
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A fast method for obtaining highly pure recombinant herpes simplex virus type 1 thymidine kinase.
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Authors
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J.Fetzer,
M.Michael,
T.Bohner,
R.Hofbauer,
G.Folkers.
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Ref.
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Protein Expr Purif, 1994,
5,
432-441.
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PubMed id
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Abstract
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Recombinant Herpes Simplex Virus Type 1 thymidine kinase (TK) was isolated in a
fast and gentle two-step procedure from Escherichia coli as a thrombin cleavable
fusion protein. The TK was expressed as an inducible glutathione S-acetyl
transferase fusion protein and purified in a first step by glutathione affinity
chromatography. Proteolytic cleavage of the column bound TK with thrombin led to
a truncated enzyme, resulting from two new and hitherto unknown cleavage sites,
determined by N-terminal sequencing. In a second step, the TK was further
purified from the cleavage products by ATP affinity chromatography, yielding
homogeneously pure TK as shown by SDS-PAGE and mass spectrometry. Both the
fusion protein and the purified enzyme show enzymatic activity with the same Km
value of 0.2 microM for the natural substrate thymidine. Determination of the
native molecular weight indicated that the pure enzyme and the fusion protein
are biologically active as homodimers. Therefore the recombinant enzyme has the
same biochemical characteristics as the viral TK, expressed in infected cells.
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