Journal
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
Volume 39, Issue 1-3, Pages 141-150Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.ijbiomac.2006.03.022
Keywords
carcinoembryonic antigen; CEA; Pichia pastoris; IMAC; concanavalin A; endoglycosydase H; scFv
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Carcinoembryonic antigen (CEA) is a seven domain membrane glycoprotein widely used as a tumour marker for adenocarcinomas and as a target for antibody-directed therapies. Structural models have proposed that the first two domains of CEA (the N terminal and adjoining A] domains) bind MFE-23, a single chain Fv antibody in experimental clinical use. We aimed to produce recombinant N-Al to test this hypothesis. The N-Al domains were expressed as soluble protein with a C-terminal hexahistidine tag (HiS(6)-tag) in the yeast Pichia pastoris. HiS(6)-tagged N-Al was captured from the supernatant by batch purification with copper-loaded Streamline (TM) Chelating, an immobilised metal affinity chromatography IMAC) matrix usually utilised in expanded bed techniques. Purified N-Al was heterogeneous with a molecular weight range from 38 to 188 kDa. Deglycosylation with endoglycosidase H (Endo H) resulted in three discrete molecular weight forms of N-Al, one partially mannosylated, one fully Endo H-digested and one fully Endo H-digested but lacking the HiS6-tag. These were separated by concanavalin A chromatography followed by HiTrap (TM) IMAC. The procedure resulted in single-band-purity, mannose-free N-Al. The binding interaction of MFE-23 to N-Al was analysed by surface plasmon resonance. The affinity constants retrieved were KD = 4.49 x 10(-9) M for the P pastoris expressed, native N-Al, and 5.33 x 10(-9) M for the Endo H-treated N-Al. To our knowledge this is the first time that two consecutive domains of CEA have been stably expressed and purified from P. pastoris. This work confirms that the CEA epitope recognised by MFE-23 resides in N-Al. (c) 2006 Elsevier B.V. All rights reserved.
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