Production of soluble and active transferrin receptor-targeting single-chain antibody using Saccharomyces cerevisiae.
OBJECTIVE
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).
Hackel
Benjamin J
BJ
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, Wisconsin 53706, USA.
Huang
Dagang
D
Bubolz
Jennifer C
JC
Wang
Xin X
XX
Shusta
Eric V
EV
D000818:Animals; D019138:Antibodies, Blocking / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology; D001812:Blood-Brain Barrier / Q000187:drug effects; D002460:Cell Line; D016503:Drug Delivery Systems; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D010957:Plasmids / Q000235:genetics; D051381:Rats; D011990:Receptors, Transferrin / Q000037:antagonists & inhibitors*; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D019138:Antibodies, Blocking; D011990:Receptors, Transferrin