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A new derivatizing agent, trimethylammoniopropyl methanethiosulphonate, is efficient for preparation of recombinant brain‐derived neurotrophic factor from inclusion bodies
Author(s) -
Inoue Makoto,
Akimaru Jiro,
Nishikawa Toyohiro,
Seki Noriaki,
Yamada Hidenori
Publication year - 1998
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1998.tb00531.x
Subject(s) - derivatization , chemistry , dithiothreitol , amine gas treating , cysteine , inclusion bodies , chromatography , neurotrophic factors , urea , brain derived neurotrophic factor , recombinant dna , organic chemistry , biochemistry , high performance liquid chromatography , enzyme , receptor , gene
Derivatization with trimethylammoniopropyl methanethiosulphonate (TAPS‐sulphonate) enabled brain‐derived neurotrophic factor (BDNF) to be prepared efficiently from Escherichia coli inclusion bodies. Reduced BDNF obtained from inclusion bodies solubilized by urea and reduced by dithiothreitol was suggested to form a complex with itself or with other compounds such as lipids. It could hardly be adsorbed on to cation‐exchange resin for partial purification prior to a refolding reaction. Reversible derivatization of cysteine residues was tested as a method of dissociating BDNF from such complexes. However, even if a methyl or aminoethyl group was introduced, BDNF could not be dissociated readily. Derivatization with TAPS‐sulphonate brought about good dissociation of BDNF, and more than 50% adsorbed on to the cation‐exchange resin. BDNF derivatized with TAPS‐sulphonate refolded well, and the refolded samples showed the same biological activity as purified BDNF. Derivatization with TAPS‐sulphonate would increase the intermolecular repulsion of BDNF, due to the positively charged character of the quaternized amine, and inhibit complex formation. Thus, TAPS‐sulphonate is effective for the preparation of BDNF under denatured conditions.