title: Refolding single-chain antibody (scFv) using lauroyl-L-glutamate as a solubilization detergent and arginine as a refolding additive.
abstract: Therapeutic potential of immunoconjugates has opened a new window for antibody-based biopharmaceuticals. Greater tissue penetration and hence enhanced cell toxicity are obtained with a smaller version of antibodies. While the whole antibody can be readily produced via mammalian expression system, antibody fragments often require refolding of insoluble proteins. Here we report a new refolding method for antibody fragments using a novel amino acid-based detergent as a solubilizing agent and arginine-assisted refolding. Inclusion bodies of antibody fragments were solubilized by 2.5% lauroyl-L-Glu (C12-L-Glu) and successfully refolded by multi-step dilution into a buffer solution containing arginine hydrochloride and thiol/disulfide-exchange reagents. Adjustment of temperature was found to be critical for increase in the refolding yield. Although each protein requires appropriate optimization, solubilization by C12-L-Glu and dilution refolding assisted by arginine can generate the native, functional antibody fragments. The procedure should enable us to utilize bacterial expression systems for the large-scale manufacturing.
authors.affiliation: Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8562, Japan.
authors.forename: Motonori
authors.initials: M
authors.lastname: Kudou
authors.forename: Daisuke
authors.initials: D
authors.lastname: Ejima
authors.forename: Haruna
authors.initials: H
authors.lastname: Sato
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Yumioka
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Arakawa
authors.forename: Kouhei
authors.initials: K
authors.lastname: Tsumoto
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry*; D002645:Chickens; D002850:Chromatography, Gel; D003902:Detergents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry; D018698:Glutamic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D002479:Inclusion Bodies; D007848:Laurates / Q000737:chemistry*; D008956:Models, Chemical; D009113:Muramidase / Q000737:chemistry; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D003902:Detergents; D007848:Laurates; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D018698:Glutamic Acid; D001120:Arginine; C421864:hen egg lysozyme; D009113:Muramidase; D019793:Fluorescein