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Expression and purification of recombinant human annexin A2 in Pichia pastoris and utility of expression product for detecting annexin A2 antibody

Paper ID Volume ID Publish Year Pages File Format Full-Text
22385 43273 2006 8 PDF Available
Title
Expression and purification of recombinant human annexin A2 in Pichia pastoris and utility of expression product for detecting annexin A2 antibody
Abstract

Annexin A2, a Ca2+-dependent phospholipid binding protein, is abundantly expressed in various human organs, which exists as either a membrane-associated, cytosolic or soluble form in serum. We constructed expression systems for recombinant human annexin A2 (rhA2) using Pichia pastoris. The systems are designed to secrete rhA2 as either the N- or C-terminally His6-tagged form to facilitate purification. Both types of rhA2 were overexpressed, but in the N-terminal-truncated form as revealed from the results of N-terminal amino acid sequencing and Western blotting. Therefore, further purification of N-terminally His6-tagged rhA2 was not feasible because of the removal of the N-terminal His6-tag sequence. C-terminally His6-tagged rhA2 was expressed as either a glycosylated or a nonglycosylated form, and the nonglycosylated form was purified using the combination of nickel-immobilized affinity, concanavalin A and cation exchanged column chromatographies. The solid-phase binding of rhA2 was examined by enzyme-linked immunosorbent assay (ELISA), which revealed the specific reactivity of rhA2 against an anti-annexin A2 monoclonal antibody. These results suggest that the expression system using P. pastoris is useful for the preparation of rhA2 that is applicable to the ELISA detection of the anti-annexin A2 antibody.

Keywords
annexin A2; Pichia pastoris; enzyme-linked immunosorbent assay; antibody detection
First Page Preview
Expression and purification of recombinant human annexin A2 in Pichia pastoris and utility of expression product for detecting annexin A2 antibody
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 101, Issue 2, February 2006, Pages 190–197
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering