A site-specific bifunctional protein labeling system for affinity and fluorescent analysis.
Most covalent protein labeling schemes require a choice between visual and affinity properties, requiring the use of multiple fusion systems where both attributes are needed. While not disruptive at the single experiment level, this detail becomes critical when addressing high-throughput experimentation. Here we develop a uniform site-specific protein tag for use in both fluorescent and affinity screening. Covalent protein tagging with a stilbene reporter via promiscuous phosphopantetheinyltransferase (PPTase) modification enables a switchable, antibody-elicited fluorescent response in solution or on affinity resin. For demonstration purposes, VibB, a natural fusion protein harboring a carrier protein domain, was labeled with a stilbene tag through PPTase modification with a stilbene-labeled coenzyme A analogue. Analysis of the resulting stilbene-tagged VibB was accomplished by fluorescent and Western blot analysis with anti-stilbene monoclonal antibody EP2-19G2. The illustration of this method for general application to fusion protein analysis offers a dual role in assisting both solution-based fluorescent analysis and surface-based affinity detection and purification.
Shute
Thomas S
TS
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358, USA.
Matsushita
Masayuki
M
Dickerson
Tobin J
TJ
La Clair
James J
JJ
Janda
Kim D
KD
Burkart
Michael D
MD
D000345:Affinity Labels / Q000737:chemistry*; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D005456:Fluorescent Dyes / Q000737:chemistry*; D007118:Immunoassay / Q000379:methods; D008722:Methods; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D013267:Stilbenes / Q000276:immunology; D017855:Transferases (Other Substituted Phosphate Groups)
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D011506:Proteins; D013267:Stilbenes; C082662:phosphopantetheinyl transferase; D017855:Transferases (Other Substituted Phosphate Groups)