Inclusion bodies accumulated in Escherichia coli are widely used for large-scale production of therapeutic proteins. Isolation and characterization of anti-Tn antigen 4E10 and anti-lacto-N-fucopentaose III 1F12 scFvs from a phage library were previously described [Yuasa et al. J. Biol. Chem. 285, 30587, 2010; Sakai et al. J Biochem. 147, 809, 2010]. Here, two scFv proteins purified from inclusion bodies were refolded using lauroyl-L-glutamate, a novel amino acid�based detergent [Kodou et al. Protein Expr Purif. 75, 46, 2011]. Inclusion bodies isolated from pET/4E10 or pET/1F12 transformed-bacterial cells were solubilized with 2.5% lauroyl-L-glutamate and subjected to reconstitution by multi-step dilution procedures into a buffer solution containing arginine and thiol/disulfide-exchange reagents. Approximately 70% were recovered as soluble proteins, which indicated that refolding in the presence of lauroyl-L-glutamate is superior to that done in the presence of guanidine hydrochloride. Determination of affinity/specificity of the refolded scFv proteins is in progress.

Expression, purification, and refolding of single chain antibodies (scFvs) from inclusion bodies

244th National Meeting (2012)

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