Identification and repair of positive binding antibodies containing randomly generated amber codons from synthetic phage display libraries

Warren D. Marcus, Stuart Lindsay, Michael Sierks

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Phage display technology allows for the rapid isolation and characterization of monoclonal antibodies that have vast potential for therapeutic and diagnostic applications. However, the panning process, which utilizes a host strain that suppresses termination by the amber codon, has an inherent bias toward clones containing randomly generated amber stop codons, complicating identification of positive binding antibodies when the antibody genes are finally expressed in a nonsupressor host. Here, we perform biopanning against a Histone 2A peptide using streptavidin- or anti-biotin-coated beads. After four rounds, a dominant clone is characterized but contains a spurious amber stop codon. A protocol is given that readily corrects the amber codon, allowing for soluble antibody production once the phagemid is transformed into a nonsuppressor bacterial strain. This work also highlights the ability to isolate antibodies against a protein antigen by using only a small peptide (15 amino acids) representing a portion of the antigen.

Original languageEnglish (US)
Pages (from-to)919-922
Number of pages4
JournalBiotechnology Progress
Volume22
Issue number3
DOIs
StatePublished - May 2006

Fingerprint

Terminator Codon
stop codon
bacteriophages
Bacteriophages
Libraries
antibodies
Antibodies
peptides
clones
antigens
host strains
streptavidin
Clone Cells
antibody formation
biotin
Antigens
Peptides
histones
Streptavidin
monoclonal antibodies

ASJC Scopus subject areas

  • Food Science
  • Biotechnology
  • Microbiology

Cite this

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