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Biotechnology and Applied Biochemistry (1998) 28, (207–213) (Printed in Great Britain)

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A new derivatizing agent, trimethylammoniopropyl methanethiosulphonate, is efficient for preparation of recombinant brain-derived neurotrophic factor from inclusion bodies

Makoto Inoue*¹, Jiro Akimaru*, Toyohiro Nishikawa*, Noriaki Seki* and Hidenori Yamada†

*Sumitomo Pharmaceuticals Research Center, 1-98, Kasugadenaka 3-chome, Konohana-ku, Osaka 554-0022, Japan, and †Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima, Okayama 700-0082, Japan

¹ To whom correspondence should be addressed.

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.

Received 18 March 1998/11 June 1998; accepted 16 June 1998

Portland Press Ltd © 1998