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Single step purification of biologically active recombinant rat basic fibroblast growth factor by immobilized metal
M Kroiher1, S Raffioni, R E Steele
1Department of Biological Chemistry, University of California, Irvine 92717-1700, USA.
Abstract:
The construction and use of a plasmid which allows the expression and single step purification of recombinant rat basic fibroblast growth factor (bFGF) is described. A cDNA encoding rat bFGF was subcloned into the expression plasmid pQE-9 (Qiagen) in such a way that the bFGF which is produced from the resulting construct contains 6 histidine residues near the amino terminus. The resulting plasmid, pQE-9-bFGF, was expressed in the E. coli strain M15[pREP4] and the 6 x His-bFGF was purified to homogeneity from the soluble fraction of the bacterial cell lysate in a single step by affinity chromatography on a nickel chelate resin. About 5 mg of 6 x His-bFGF was obtained from the soluble fraction from one liter of bacterial cell culture. Testing of the 6 x His-bFGF in a PC12 cell differentiation assay showed that its activity was comparable to the activities for native bFGF and recombinant bFGF purified by multistep methods.