Journal
BIOPROCESS AND BIOSYSTEMS ENGINEERING
Volume 31, Issue 6, Pages 559-568Publisher
SPRINGER
DOI: 10.1007/s00449-008-0203-y
Keywords
Pichia pastoris; single-chain; fluorophore; enzyme; recombinant fusion protein; fermentation; purification
Funding
- Ernst-von-Leyden Stipendium of the Berliner Krebsgesellschaft
- Deutsche Krebshilfe [1072981]
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Recombinant antibody fusion constructs with heterologous functional domains are a promising approach to new therapeutic targeting strategies. However, expression of such constructs is mostly limited to cost and labor-intensive mammalian expression systems. Here we report on the employment of Pichia pastoris for the expression of heterologous antibody fusion constructs with green fluorescent protein, A33scFv::GFP, or with cytosine deaminase, A33scFv::CDy, their production in a biofermenter and a modified purification strategy. Combined, these approaches improved production yields by about thirty times over established standard protocols, with extracellular secretion of the fusion construct reaching 12.0 mg/l. Bifunctional activity of the fusion proteins was demonstrated by flow cytometry and an in-vitro cytotoxicity assay. With equal amounts of purified protein, the modified purification method lead to higher functional results. Our results demonstrate the suitability of methylotrophic Pichia expression systems and laboratory-scale bioreactors for the production of high quantities of bifunctionally active heterologous single-chain fusion proteins.
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