MODIFICATION OF AN ANTI-CTLA4 ELASTIN-LIKE POLYPEPTIDE FUSION USING PCR-MEDIATED SITE-DIRECTED DELETION

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2024-06-08

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University of Southern California

Abstract

Immune checkpoint inhibitors have gained increasing interest in cancer immunotherapy following their great clinical success. The first approved agent in this class targets Cytotoxic T- lymphocyte-associated protein-4 (CTLA-4). Our group genetically engineered a fusion protein combining an antibody fragment targeting CTLA-4 with an elastin-like polypeptide (ELP). Unlike other scFv-ELP fusions we worked with, this construct is expressed in low yield (2.88 ± 2.13 mg/L culture). To enhance protein expression in the soluble fragment, we modified the DNA sequence of the construct using polymerase chain reaction (PCR) in a method known as site-directed mutagenesis. The newly synthesized plasmid was verified by DNA sequencing, which revealed an in-situ deletion. The modified construct was successfully expressed in E. coli, resulting in a 17- fold increase in protein yield. To explore the impact of this manipulation on the biophysical properties of the proteins, we analyzed the molecular weight, nanoparticle size, and thermal phase behavior. This PCR-mediated mutation protocol is simple, effective, and may enable future modifications of plasmid fragments with reagents available for basic recombinant DNA settings.

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PCR-mediated mutation, polymerase chain reaction, PCR, elastin-like polypeptide, ELP, Recombinant DNA technology, Plasmid

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