Summary
Numerous techniques are available for investigating protein-ligand interactions. The phage display technique is one such method routinely used to identify antibody-antigen interactions and has the benefit of being easily adaptable to high-throughput screening platforms. Once identified, antigen-binding domains on fragment antibodies or single-chain fragment antibodies (scFv) can be expressed and purified for further studies. In this chapter, we describe a method for high-level expression of a phage display-derived scFv in Pichia pastoris. The phage display-derived antibody A33scFv recognizes a cell surface glycoprotein (designated A33) expressed in colon cancer that serves as a target antigen for radioimmunoimaging and/or immunotherapy of human colon cancer. The expression and purification of A33scFv was optimized for the methylotrophic yeast P. pastoris. P. pastoris with a MutS phenotype was selected to express A33scFv under regulation of the methanol-inducible AOX1 promoter. Here we describe a large-scale fed-batch fermentation process with an efficient online closed-loop methanol control for the production of the recombinant protein. Purification of A33scFv from clarified culture medium was done using a two-step chromatographic procedure using anion exchange and hydrophobic interaction chromatography, resulting in a final product with more than 90% purity. This chapter provides protocols that can be used as a base for process development of recombinant protein expression in P. pastoris and purification of these proteins for use in further functionality studies and in diagnostic and therapeutic applications.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Damasceno, L.M., Lee, F., Ritter, G., Old, L., Batt, C. (2009). High-Level Expression of a Phage Display-Derived scFv in Pichia pastoris . In: Aitken, R. (eds) Antibody Phage Display. Methods in Molecular Biology, vol 562. Humana Press. https://doi.org/10.1007/978-1-60327-302-2_18
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DOI: https://doi.org/10.1007/978-1-60327-302-2_18
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