Purpose. This study describes the soluble production, purification, and functional testing of an anti-transferrin receptor single-chain antibody (OX26 scFv) using the yeast Saccharomyces cerevisiae. Methods. The yeast secretion apparatus was optimized by modulating expression temperature, the folding environment of the endoplasmic reticulum, and gene dosage. Secreted scFv was purified using immobilized metal affinity chromatography, and tested for binding and internalization into the RBE4 rat brain endothelial cell line. Results. Secretion of OX26 scFv was optimal when expression was induced at 20°C. Co-overexpression of heavy chain binding protein and protein disulfide isomerase elevated scFv expression levels by 10.4 ± 0.3-fold. Optimization of scFv gene dosage increased secretion by 7.1 ± 0.2-fold, but the overall benefits of binding protein and protein disulfide isomerase overexpression were diminished. Purified OX26 scFv yields of 0.5 mg/L secreted protein were achieved, and the scFv was actively internalized into RBE4 cells with a pattern similar to that observed with intact OX26 monoclonal antibody. Conclusions. The optimized S. cerevisiae expression system is amenable to production of soluble and active brain targeting OX26 scFv, and the yeast-produced scFv has potential for the targeting and delivery of small molecules, proteins, or drug carriers across the blood-brain barrier(BBB).
Bibliographical noteFunding Information:
Special thanks are extended to Grzegorz Sabat, James Brown, and Dr. Amy Harms of the University of Wisconsin Biotechnology Center for their assistance in analyzing peptide mapping and intact protein molecular weight measurement data. Thanks are also due to Michael Pereckas at the Medical College of Wisconsin for his assistance with protein sequencing. This study was funded by the Whitaker Foundation award RG-02-0077 and NSF CAREER award BES-0238864. B.J. Hackel was an NSF REU recipient and a University of Wisconsin Hilldale undergraduate research fellow.
- Blood-brain barrier
- Drug targeting
- Single-chain antibody