Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments.
We have produced single-chain antibody fragments (scFv) in Saccharomyces cerevisiae at levels up to 20 mg/L in shake flask culture by a combination of expression level tuning and overexpression of folding assistants. Overexpression of the chaperone BiP or protein disulfide isomerase (PDI) increases secretion titers 2-8 fold for five scFvs. The increases occur for scFv expression levels ranging from low copy to ER-saturating overexpression. The disulfide isomerase activity of PDI, rather than its chaperone activity, is responsible for the secretion increases. A synergistic increase in scFv production occurs upon cooverexpression of BiP and PDI.
Department of Chemical Engineering, University of Illinois, Urbana 61801, USA.
E V
EV
Shusta
R T
RT
Raines
A
A
Plückthun
K D
KD
Wittrup
D019943:Amino Acid Substitution; D000906:Antibodies; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002846:Chromatography, Affinity; D003545:Cysteine; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D018628:Gene Dosage; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006360:Heat-Shock Proteins; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; C069242:McPC603 antibody; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D019704:Protein Disulfide-Isomerases; D003545:Cysteine