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Journal of Biochemistry Advance Access published online on March 23, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm075
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© 2007 The Japanese Biochemical Society

Stable supply of large amounts of human Fab from the inclusion bodies in E. coli.

Testuro Fujii*, Takatoshi Ohkuri*, Reiko Onodera and Tadashi Ueda{dagger}

Department of Immunology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan

{dagger}Corresponding author; Tadashi Ueda, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. TEL, +81-92-642-6662; FAX, +81-92-642-6667; E-mail: ueda{at}phar.kyushu-u.ac.jp

Received November 28, 2006; Accepted February 25, 2007


  Abstract

Recombinant human Fab-H chain and L chain were separately expressed as inclusion body using E. coli. After solubilization of Fab-H chain and L-chain by the reduction and S-alkyldisulphidationalkylation in 8 M urea, about 100 mg of purified Fab-H chain and about 160 mg of L chain could be obtained from 1L of each culture by ion-exchange chromatogram in the presence of 8M urea. Combination of the lyophilized Fab-H chain and L chain could be efficiently folded to native human Fab by using the stepwise dialysis method and the human Fab was purified with cation-exchange chromatogram. In the folding procedure, it was found that cysteamine and cystamine with positive charge were effective to improve the folding yield of human Fab. Moreover, from comparison of folding yield in the presence of ten kinds of additives, it was suggested that taurine was effective to improve the folding of human Fab. Consequently, we could obtain about 60 mg of folded human Fab from 1 L of each culture under the optimum conditions.

Key Words: human Fab, folding, rheumatoid factor, taurine

* Equally contributing authors


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