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Evolution of a carbohydrate binding module into a protein-specific binder

Paper ID Volume ID Publish Year Pages File Format Full-Text
14180 1180 2006 7 PDF Available
Title
Evolution of a carbohydrate binding module into a protein-specific binder
Abstract

A carbohydrate binding module, CBM4-2, derived from the xylanase (Xyn 10A) of Rhodothermus marinus has been used as a scaffold for molecular diversification. Its binding specificity has been evolved to recognise a quite different target, a human monoclonal IgG4. In order to understand the basis for this drastic change in specificity we have further investigated the target recognition of the IgG4-specific CBMs. Firstly, we defined that the structure target recognised by the selected CBM-variants was the protein and not the carbohydrates attached to the glycoprotein. We also identified key residues involved in the new specificity and/or responsible for the swap in specificity, from xylan to human IgG4. Specific changes present in all these CBMs included mutations not introduced in the design of the library from which the specific clones were selected. Reversion of such mutations led to a complete loss of binding to the target molecule, suggesting that they are critical for the recognition of human IgG4. Together with the mutations introduced at will, they had transformed the CBM scaffold into a protein binder. We have thus shown that the scaffold of CBM4-2 is able to harbour molecular recognition for either carbohydrate or protein structures.

Keywords
AE, affinity electrophoresis; BSA, bovine serum albumin; CBM, carbohydrate binding module; ConA, concanavalin A; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; IPTG, isopropyl-β-d-thiogalactoside; PBS, phosphate-buffered saline; PC
First Page Preview
Evolution of a carbohydrate binding module into a protein-specific binder
Publisher
Database: Elsevier - ScienceDirect
Journal: Biomolecular Engineering - Volume 23, Issues 2–3, June 2006, Pages 111–117
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering