title: Baculovirus expression of human basic fibroblast growth factor from a synthetic gene: role of the Kozak consensus and comparison with bacterial expression.
abstract: 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.
authors.affiliation: Biophysics Laboratories, University of Portsmouth, UK.
authors.forename: D
authors.initials: D
authors.lastname: Hills
authors.forename: C
authors.initials: C
authors.lastname: Crane-Robinson
mesh_terms: 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
chemical_list: D004274:DNA, Recombinant; D008934:Mitogens; D016222:Fibroblast Growth Factor 2