Overproduction of biologically-active human nerve growth factor in Escherichia coli.
A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E. coli. The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the beta-lactamase signal peptide. The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene. E. coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the beta-lactamase signal peptide. The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth. This biological activity was comparable to that of authentic mouse NGF. E. coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein. Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells.
Advanced Research Laboratory, Hitachi Ltd., Saitama, Japan.
K
K
Fujimori
S
S
Fukuzono
N
N
Kotomura
N
N
Kuno
N
N
Shimizu
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002499:Centrifugation, Density Gradient / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D005813:Genes, Synthetic; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D009838:Oligodeoxyribonucleotides; D016716:PC12 Cells; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping
D009414:Nerve Growth Factors; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins