High-throughput screening for soluble recombinant expressed kinases in Escherichia coli and insect cells.
We have constructed a dual expression vector for the production of recombinant proteins in both Escherichia coli and insect cells. In this vector, the baculoviral polyhedrin promoter was positioned upstream of the bacteriophage T7 promoter and the lac operator. This vector, designated pBEV, was specifically designed to exploit the advantages that both hosts would provide. This vector also facilitates one-stop cloning, thereby simplifying the expression process for automation, and the development of a high-throughput method for protein expression. Utilizing the multi-system vector pBEV, a high-throughput process was developed with expression in deep-well blocks and purification in micro-titer plates enabling the identification of expression and solubility in both E. coli and insect cells. In this study, using pBEV, we have successfully expressed and purified multiple human kinases produced in E. coli and insect cells. Our results validate expression screening as a strategy to rapidly triage proteins identifying the optimum expression system and conditions for production.
Chambers
Stephen P
SP
Vertex Pharmaceuticals Inc., 130 Waverly Street, Cambridge, MA 02139-4242, USA. chambers@vrtx.com
Austen
Douglas A
DA
Fulghum
John R
JR
Kim
Walter M
WM
D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D002460:Cell Line; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007313:Insecta / Q000166:cytology*; D007763:Lac Operon / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011494:Protein Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D000099270:Y-Family DNA Polymerases
D004259:DNA-Directed DNA Polymerase; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases