Stable supply of large amounts of human Fab from the inclusion bodies in E. coli.
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.
Fujii
Testuro
T
Department of Immunology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Ohkuri
Takatoshi
T
Onodera
Reiko
R
Ueda
Tadashi
T
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003538:Cystamine / Q000494:pharmacology; D003543:Cysteamine / Q000494:pharmacology; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis; D002479:Inclusion Bodies / Q000378:metabolism*; D017510:Protein Folding; D011817:Rabbits
D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D003543:Cysteamine; D003538:Cystamine