Expression and purification of histidine-tagged bacteriophage T7 DNA polymerase.

Abstract:

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.

Polymerases:

T7

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