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Journal of Biochemistry Advance Access originally published online on March 23, 2007
Journal of Biochemistry 2007 141(5):699-707; 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}To whom correspondence should be addressed. 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 Escherichia coli. After solubilization of Fab-H chain and L chain by the reduction and S-alkyldisulphidation in 8 M urea, about 100 mg of purified Fab-H chain and about 160 mg of L chain could be obtained from 1 l of each culture by ion-exchange chromatogram in the presence of 8 M 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: folding, human Fab, rheumatoid factor, taurine

Abbreviations: CTA, cystamine; CTE, cysteamine; DDP, 3,3’-dithiodipropionic acid; GSSG, oxidized glutathione; ME, mercaptoethanol; MP, mercaptopropionic acid; RF, rheumatoid factors; scFv, single-chain Fv

*The first two authors have contributed equally to this work.


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