A new derivatizing agent, trimethylammoniopropyl methanethiosulphonate, is efficient for preparation of recombinant brain-derived neurotrophic factor from inclusion bodies.
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.
Inoue
M
M
Sumitomo Pharmaceuticals Research Center, 1-98, Kasugadenaka 3-chome, Konohana-ku, Osaka 554-0022, Japan.
Akimaru
J
J
Nishikawa
T
T
Seki
N
N
Yamada
H
H
D019208:Brain-Derived Neurotrophic Factor / Q000737:chemistry* / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000737:chemistry / Q000648:ultrastructure; D002479:Inclusion Bodies / Q000737:chemistry*; D008698:Mesylates / Q000737:chemistry*; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D019208:Brain-Derived Neurotrophic Factor; D008698:Mesylates; D011994:Recombinant Proteins; C117592:trimethylammoniopropyl methanethiosulfonate