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Construction of an agglutination tool: recombinant Fab fragments biotinylated in vitro

Paper ID Volume ID Publish Year Pages File Format Full-Text
33700 44988 2009 7 PDF Available
Title
Construction of an agglutination tool: recombinant Fab fragments biotinylated in vitro
Abstract

The pComb3H vector system is used for constructing and panning recombinant antibody libraries. It allows for expression of monovalent Fab fragments, either on the surface of M13 phage, or in the form of soluble proteins secreted into the periplasmic space of bacteria. We constructed a modified pComb3H vector containing cDNA encoding for a 23-amino acid fragment of the Escherichia coli biotin carboxy carrier protein (BCCP), which is an acceptor sequence for biotinylation. The vector was used to express the Fab fragment recognizing human glycophorin A. The purified Fab fragment containing this biotin acceptor sequence was effectively biotinylated in vitro using biotin ligase (BirA). The specificity and avidity of the biotinylated Fab fragments were similar to the previously produced, unmodified Fab fragments. An avidin–alkaline phosphatase conjugate was used to detect the recombinant Fab fragments, instead of secondary antibody. In addition, when biotinylated Fab fragments were mixed with avidin, red blood cells were directly agglutinated.

First Page Preview
Construction of an agglutination tool: recombinant Fab fragments biotinylated in vitro
Publisher
Database: Elsevier - ScienceDirect
Journal: New Biotechnology - Volume 26, Issue 5, 30 November 2009, Pages 215–221
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering