Preparation of biologically active single-chain variable antibody fragments that target the HIV-1 gp120 V3 loop
Corresponding Author(s) : S. G. Sarafianos
Cellular and Molecular Biology,
Vol. 58 No. 1: Frontiers in biological sciences issue
KD-247 is a humanized monoclonal antibody that targets the third hypervariable (V3) loop of gp120. It can efficiently neutralize a broad panel of clade B, but not non-clade B, HIV-1 isolates. To overcome this limitation, we are seeking to prepare genetically-engineered single-chain variable fragments (scFvs) of KD-247 that will have broader neutralizing activity against both clade B and non-clade B HIV-1 isolates. Initial attempts of optimizing the expression of KD-247 scFv have resulted in the formation of insoluble protein. Therefore, we have established purification protocols to recover, purify, and refold the KD-247 scFv from inclusion bodies. The protocol involved step-wise refolding of denatured scFv by dilution, dialysis, and on-column nickel-affinity purification. Monomeric scFv was further purified by size-exclusion chromatography. Using far UV circular dichroism (CD) spectroscopy we confirmed the expected beta-sheet profile of the refolded KD-247 scFv. Importantly, the refolded KD-247 scFv showed neutralizing activity against replication-competent HIV-1 BaL and JR-FL Env pseudotyped HIV-1, at potency comparable to that of the native full-size KD-247 antibody. Ongoing studies focus on the application of this system in generating KD-247 scFv variants with the ability to neutralize clade B and non-clade B HIV-1 isolates.
KD-247 neutralizing antibody HIV gp120 V3 loop scFv purification refolding.
Ong, Y. T., Kirby, K. A., Hachiya, A., Chiang, L. A., Marchand, B., Yoshimura, K., Murakami, T., Singh, K., Matsushita, S., & Sarafianos, S. G. (2012). Preparation of biologically active single-chain variable antibody fragments that target the HIV-1 gp120 V3 loop. Cellular and Molecular Biology, 58(1), 71–79. Retrieved from https://cellmolbiol.org/index.php/CMB/article/view/580
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