Baculovirus expression of human basic fibroblast growth factor from a synthetic gene: role of the Kozak consensus and comparison with bacterial expression.
Synthetic genes encoding the 146 and 155 amino acid forms of human basic fibroblast growth factor (bFGF) were constructed with codon usage biased towards the polyhedrin-encoding gene of Autographa californica nuclear polyhedrosis virus (AcNPV). Expression of both bFGF genes in Spodoptera frugiperda (SF-21) suspension cell culture using a recombinant baculovirus yielded approximately 2.5 mg of mitogenically fully active protein per 10(9) cells following heparin-affinity chromatography. To improve translational efficiency, the Kozak consensus sequence was introduced and it was found that neither the replacement of a pyrimidine by a purine at position -3, nor the nature of the base at position +4 had any noticeable effect on the final levels of bFGF expression in SF-21 cells. The bases at these critical points in the consensus do not therefore play a major role in expression levels of the bFGF synthetic genes. The two synthetic genes were also expressed in Escherichia coli as native proteins using the T7 expression system. 5 mg of mitogenically fully active bFGF were obtained from 1 l of bacterial culture. Both insect cell- and E. coli-derived bFGF were equally mitogenic for Swiss 3T3 fibroblasts.
Hills
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Biophysics Laboratories, University of Portsmouth, UK.
Crane-Robinson
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D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D016222:Fibroblast Growth Factor 2 / Q000235:genetics* / Q000502:physiology; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008934:Mitogens / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera
D004274:DNA, Recombinant; D008934:Mitogens; D016222:Fibroblast Growth Factor 2