Production of soluble and active transferrin receptor-targeting single-chain antibody using Saccharomyces cerevisiae

Benjamin J. Hackel, Dagang Huang, Jennifer C. Bubolz, Xin X. Wang, Eric V. Shusta

Research output: Contribution to journalArticlepeer-review

45 Scopus citations

Abstract

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).

Original languageEnglish (US)
Pages (from-to)790-797
Number of pages8
JournalPharmaceutical research
Volume23
Issue number4
DOIs
StatePublished - Apr 1 2006

Keywords

  • Blood-brain barrier
  • Drug targeting
  • Single-chain antibody
  • Yeast

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