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In vitro molecular evolution of AL NEIBMs improved immunoglobulin (Ig) binding and antibody detection

Paper ID Volume ID Publish Year Pages File Format Full-Text
23202 43419 2014 10 PDF Available
Title
In vitro molecular evolution of AL NEIBMs improved immunoglobulin (Ig) binding and antibody detection
Abstract

•We constructed four combinatorial phage libraries displaying AL mutants with random mutations at different amino acid positions in the A domain of NEIBMs.•We examine the binding activity through human IgM-directed in vitro evolution.•Two AL mutants showed significantly improved IgM and IgG binding activity.•These AL mutants could substantially contribute to the use of AL in antibody detection.•This study provides an example of successful protein engineering through in vitro molecular evolution.

AL (SpA A domain-PpL B3 domain), LD5 (PpL B3 domain-SpA D domain-PpL B3 domain-SpA D domain-PpL B3 domain, L-D-L-D-L) and LD3 (PpL B3 domain-SpA D domain-PpL B3 domain, L-D-L) are novel evolved Ig binding molecules (NEIBMs) derived from the in vitro molecular evolution of combinatorial phage libraries displaying randomly rearranged Ig-binding domains of protein A and protein L. These molecules all showed novel Ig-binding properties of double-site binding to the VH3 and Vκ regions of human Ig Fab and high affinity for human IgM, which enhanced IgM detection in the anti-HCV ELISA assay. In this double-site binding, the A domain binds to the VH3 chain with low affinity. Whether the appropriate mutations in the A domain could improve this binding remains unknown. In this study, four combinatorial phage libraries displaying AL mutants with random mutations at different amino acid positions in the A domain were constructed. Seven AL mutant phages with significantly improved Ig binding activity were obtained from the phage library displaying AL mutants randomly mutated at positions 27 and 34 through human IgM-directed in vitro evolution. Two of the seven prokaryotically expressed AL mutants, AL (VV) and AL (KA), exhibited IgM and IgG binding activities equivalent to those of wild-type AL, whereas other mutants showed attenuated binding. However, after labeling with HRP, AL (VV) and AL (KA) showed improved IgM and IgG binding activity, which significantly improved the detection in the anti-HCV assay. Thus, the present study demonstrates that the binding properties of AL were successfully improved through phage-based molecular evolution, which could substantially contribute to the use of AL in antibody detection, and provides an example of successful protein engineering through in vitro molecular evolution.

Keywords
NEIBM; Human IgM; AL mutants; Phage-based molecular evolution; Antibody detection
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In vitro molecular evolution of AL NEIBMs improved immunoglobulin (Ig) binding and antibody detection
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 184, 20 August 2014, Pages 118–127
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us