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 degrees 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).
PURPOSE: This study describes the soluble production, purification, and functional testing of an anti-transferrin receptor single-chain antibody (OX26 scFv) using the yeastSaccharomyces 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 degrees 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).
Authors: Michael J Feldhaus; Robert W Siegel; Lee K Opresko; James R Coleman; Jane M Weaver Feldhaus; Yik A Yeung; Jennifer R Cochran; Peter Heinzelman; David Colby; Jeffrey Swers; Christilyn Graff; H Steven Wiley; K Dane Wittrup Journal: Nat Biotechnol Date: 2003-01-21 Impact factor: 54.908
Authors: Carmen Wängler; Dina Nada; Georg Höfner; Simone Maschauer; Björn Wängler; Stephan Schneider; Esther Schirrmacher; Klaus T Wanner; Ralf Schirrmacher; Olaf Prante Journal: Mol Imaging Biol Date: 2011-04 Impact factor: 3.488
Authors: Benjamin J Tillotson; Iñigo F de Larrinoa; Colin A Skinner; Derek M Klavas; Eric V Shusta Journal: Protein Eng Des Sel Date: 2012-10-28 Impact factor: 1.650