Open AccessProduction of Biologically Active Mature Brain-derived Neurotrophic Factor inEscherichia coli
Author(s) -
Shinichi Fukuzono,
Kiyoshi Fujimori,
Norio Shimizu
Publication year - 1995
Publication title -
bioscience, biotechnology, and biochemistry
Language(s) - Uncategorized
Resource type - Journals
SCImago Journal Rank - 0.509
H-Index - 116
eISSN - 1347-6947
pISSN - 0916-8451
DOI - 10.1271/bbb.59.1727
Subject(s) - escherichia coli , neurotrophic factors , complementary dna , brain derived neurotrophic factor , biological activity , microbiology and biotechnology , chemistry , biology , recombinant dna , neurotrophin , gene expression , nerve growth factor , biochemistry , gene , in vitro , receptor
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.