Production of biologically active mature brain-derived neurotrophic factor in Escherichia coli.
A gene coding for a brain-derived neurotrophic factor (BDNF) obtained from rat BDNF cDNA was used to construct an expression plasmid, pTRSBDNF. It contained a BDNF gene that was fused, in frame, to a region encoding the beta-lactamase signal peptide. The E. coli HB101 harboring pTRSBDNF produced 18 mg/liter of mature protein. The E. coli cells produced a larger amount of BDNF than that of human nerve growth factor (hNGF), which was produced using the same expression system. The E. coli cells harboring pTRSBDNF were sonicated and centrifuged to obtain a supernatant and precipitate. The BDNF purified from the supernatant containing 20% of the total BDNF had high biological activity (EC50 of 30 pg/ml) against neurons of chick dorsal root ganglia. On the other hand, the BDNF purified from the precipitate had low biological activity (EC50 of 2 ng/ml) and incorrect disulfide bonds.
Advanced Research Laboratory, Hitachi, Ltd., Saitama, Japan.
S
S
Fukuzono
K
K
Fujimori
N
N
Shimizu
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019208:Brain-Derived Neurotrophic Factor; D002642:Chick Embryo; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D051379:Mice; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000096:biosynthesis / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis
D019208:Brain-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins