Refolding Technology for scFv Using a New Detergent, N-Lauroyl-L-glutamate and Arginine

Abstract

Monoclonal antibodies to the soluble antigens or cell surface markers hold great promise as effective human therapeutics. One of the major disadvantages is its large size, which prevents efficient penetration into the target tissues. Smaller version of antibodies, which has only antigen binding sites, is extensively investigated. It becomes increasingly apparent, however, that these smaller fragments of antibodies are rather difficult to produce, as the normally efficient mammalian secretion system does not work well for these fragments. Thus, refolding of insoluble proteins produced in Escherichia coli is a method of choice, although such refolding is mainly based on trial-and-error experiment. Here we describe a novel refolding system using a new amino acid-based detergent, N-lauroyl-L-glutamate, and arginine. This detergent appears to readily dissociate from proteins below critical micelle concentration (CMC), while remaining effective in protein solubilization above CMC. Arginine suppresses protein aggregation when the detergent concentration was reduced below CMC. The interaction of the detergent and arginine with proteins, which play an important role in protein refolding, will be discussed in great length.

Refolding Technologies for Antibody Fragments

“…The refolding process consists of solubilization, removal of solubilizing reagents and, for disulfide-containing proteins such as antibody fragments, oxidation of sulfhydryl groups. We have developed a novel refolding technology using an acylamino acid detergent, N-lauroyl-L-glutamate (C12-L-Glu) [12,13], and as reviewed in one of this series [14], based on two model proteins and scFv constructs. Here, we describe the various refolding technologies and compare with refolding using C12-L-Glu.…”

Sodium Dodecyl Sulfate Analogs as a Potential Molecular Biology Reagent

“…The cleaning properties of SLG have been studied because of its cosmetic and cleansing applications [16]. When compared to Sarkosyl, SLG was slightly weaker in suppressing the surface tension of water, although both detergents are strong surface tension suppressors [17]. SLG is much stronger at stabilizing the foams than Sarkosyl despite their similarity in decreasing the surface tension of water.…”