MersV1, Istex, Corpus, bibRecord, 001E82

A Fast Method for Obtaining Highly Pure Recombinant Herpes Simplex Virus Type 1 Thymidine Kinase

Identifieur interne : 001E82 ( Istex/Corpus ); précédent : 001E81; suivant : 001E83

A Fast Method for Obtaining Highly Pure Recombinant Herpes Simplex Virus Type 1 Thymidine Kinase

Auteurs : J. Fetzer ; M. Michael ; T. Bohner ; R. Hofbauer ; G. Folkers

Source :

Protein Expression and Purification [ 1046-5928 ] ; 1994.

RBID : ISTEX:ADED910A0F36F32953776918AF7CB18206782896

Abstract

Abstract: 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 μM 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.

Url:

https://api.istex.fr/ark:/67375/6H6-DMDC129P-T/fulltext.pdf

DOI: 10.1006/prep.1994.1062

Links to Exploration step

ISTEX:ADED910A0F36F32953776918AF7CB18206782896

Le document en format XML

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A Fast Method for Obtaining Highly Pure Recombinant Herpes Simplex Virus Type 1 Thymidine Kinase

A Fast Method for Obtaining Highly Pure Recombinant Herpes Simplex Virus Type 1 Thymidine Kinase

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