Expression and purification of histidine-tagged bacteriophage T7 DNA polymerase.
The formation of inclusion bodies is a frequent consequence of high-level production of foreign protein in the cytoplasm of Escherichia coli. This phenomenon is also observed with bacteriophage T7 gene 5 protein, the phage-encoded subunit of T7 DNA polymerase, if expression is based on the T5 promoter/lac operator transcription-translation system present in a vector with ColE1 origin of replication. To avoid tedious procedures for recovering protein from insoluble aggregates, we studied the expression of T7 gene 5 protein using a series of E. coli strains, and optimized the yield of soluble, histidine-tagged (His-tagged) protein by varying the respective growth conditions (temperature, amount of inducer isopropyl-beta-d-thiogalactopyranoside, and presence of organic osmolytes). Although the expression levels in three different strains (BL21, SG13009, and XL1-Blue) were almost comparable with a given set of growth conditions, the yields of soluble protein differed markedly. The largest quantities of soluble, His-tagged T7 gene 5 protein were achieved using "cloning strain" XL1-Blue which benefitted significantly from the presence of sorbitol and glycine betaine-in contrast to the expression strains BL21 and SG13009. Purification of His-tagged T7 gene 5 protein was achieved using single-step metal-affinity chromatography that yielded large amounts of highly active polymerase (97% homogeneity). The application of this expression/purification approach represents not only a useful method to purify large quantities of T7 DNA polymerase for structural investigations but also, provides a fast and efficient protocol for the parallel purification of T7 DNA polymerase variants, e.g., for automated screenings or directed evolution experiments.
Schlicke
Marina
M
Applied Molecular Evolution, Institute for Biology II, University of Leipzig, Liebigstrasse 18, D-04103 Leipzig, Germany.
Brakmann
Susanne
S
D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D005470:Fluorometry; D015870:Gene Expression; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D006639:Histidine; D004259:DNA-Directed DNA Polymerase