High-level expression of a phage display-derived scFv in Pichia pastoris.
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 Mut(S) 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.
Damasceno
Leonardo M
LM
Food Science, Cornell Unviersity, Ithaca, NY 14853, USA. lmd42@cornell.edu
Lee
Frank
F
Ritter
Gerd
G
Old
Lloyd
L
Batt
Carl
C
D019149:Bioreactors; D002852:Chromatography, Ion Exchange; D005285:Fermentation; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology* / Q000302:isolation & purification; D008562:Membrane Glycoproteins / Q000276:immunology*; D019151:Peptide Library; D010843:Pichia / Q000276:immunology*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000276:immunology* / Q000302:isolation & purification
C104258:GPA33 protein, human; D007135:Immunoglobulin Variable Region; D008562:Membrane Glycoproteins; D019151:Peptide Library; D011994:Recombinant Proteins