A fast method for obtaining highly pure recombinant herpes simplex virus type 1 thymidine kinase.
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.
Fetzer
J
J
Department of Pharmacy, Swiss Federal Institute of Technology, ETH-Zentrum, Zürich.
Michael
M
M
Bohner
T
T
Hofbauer
R
R
Folkers
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D013937:Thymidine Kinase; D013917:Thrombin; D013936:Thymidine