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Diversification of a Bacterial ADP‐Ribosyltransferase
Author(s) -
Sung Vicky,
Kallert Stephanie
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.712.1
Subject(s) - enzyme , protein engineering , mutagenesis , directed evolution , computational biology , phage display , biochemistry , directed molecular evolution , biology , chemistry , gene , mutation , mutant , peptide
OBJECTIVE To accelerate the enzyme evolution, we diversified a bacterial ADP‐ribosyltransferase, cholix toxin catalytic fragment, to modify different substrates. METHODS Previously, we identified a diffusible intermediate that could modify target residues in a proximity dependent manner (submitted in the same meeting, Topic #2278‐ASBMB‐Enzyme Mechanism, Number 540). We utilized a phage display library screening strategy to select for variants to bind to different substrates of interests, followed by enzymatic screening for the selected variants retaining enzymatic activity. Site‐directed mutagenesis, enzymatic assays and 2‐D‐electrophoresis analysis were used to detect the in vitro modification of the substrates by the engineered enzymes. RESULTS We showed several examples of engineered cholix toxin catalytic fragment variants that can modify various substrates, including enhanced green fluorescent protein (EGFP) and mouse immunoglobulins. CONCLUSION We anticipate our findings to be a starting point for more sophisticated engineering. Such enzymes can be useful tools for basic research or therapeutic applications.