Rapid isolation of homogeneous cloned T7 gene 5 protein and T7 DNA polymerase by affinity chromatography on immobilized thioredoxin.
Phage T7 DNA polymerase consists of a strong 1:1 complex of T7 gene 5 protein (80 kDa) and the reduced form of Escherichia coli thioredoxin (12 kDa). Immobilization of E. coli thioredoxin on the agarose matrix Affi-Gel retained both its redox activity and its ability to bind T7 gene 5 protein. This was used to develop a simple and fast high-yield purification method. Cloned T7 gene 5 protein, expressed in a thioredoxin-negative host cell, was isolated in pure and highly active form after elution from Affi-Gel--thioredoxin with a pH gradient from 10 to 12. This purification step separated gene 5 protein from variable amounts of two sets of reconstituting large polypeptide fragments without catalytic activity. Proteolytic cleavage in vivo probably gave rise to the fragments, the generation of which was mimicked by trypsin cleavage of pure gene 5 protein. The gene 5 protein preparation had an inherent low DNA polymerase and double-stranded 3'-exonuclease activity, which was stimulated at least 30-fold by the presence of reduced thioredoxin. Highly active and pure T7 DNA polymerase was obtained by reconstitution of gene 5 protein with thioredoxin and was isolated by phosphocellulose or FPLC Mono Q chromatography. The gene 5 protein and T7 DNA polymerase preparations are suitable for further physicochemical characterization and as reagents in DNA sequencing.
Slaby
I
I
Department of Physiological Chemistry, Karolinska Institutet, Stockholm, Sweden.
Holmgren
A
A
D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification*; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics*; D013879:Thioredoxins; D014357:Trypsin; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification*
D014764:Viral Proteins; D013879:Thioredoxins; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D014357:Trypsin