A comprehensive system for protein purification and biochemical analysis based on antibodies to c-myc peptide.
The genomics revolution has created a need for increased speed and generality for recombinant protein production systems as well as general methods for conducting biochemical assays with the purified protein products. 9E10 is a well-known high-affinity antibody that has found use in a wide variety of biochemical assays. Here we present a standardized system for purifying proteins with a simple epitope tag based on c-myc peptide using an antibody affinity column. Antibodies with binding parameters suitable for protein purification have been generated and characterized. To purify these antibodies from serum-containing medium without carrying through contaminating immunoglobulin G, a peptide-based purification process was developed. A fluorescence polarization binding assay was developed to characterize the antigen-antibody interaction. Protein purification protocols were optimized using a fluorescein-labeled peptide as a surrogate "protein." Binding and elution parameters were evaluated and optimized and basic operating conditions were defined. Several examples using this procedure for the purification of recombinant proteins are presented demonstrating the generality of the system. In all cases tested, highly pure final products are obtained in good yields. The combination of the antibodies described here and 9E10 allow for almost any biochemical application to be utilized with a single simple peptide tag.
Hillman
M C
MC
Applied Biotechnology Department, The DuPont Pharmaceuticals Company, Experimental Station Route 141 and Henry Clay Road, Wilmington, Delaware 19880-0336, USA.
Yang
L S
LS
Sun
S
S
Duke
J L
JL
O'Neil
K T
KT
Kochie
J E
JE
Karjoo
A
A
Nath
P
P
Breth
L A
LA
Murphy
K
K
Ross
O H
OH
Burn
T C
TC
Hollis
G F
GF
Wynn
R
R
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000939:Epitopes; D005260:Female; D005455:Fluorescent Antibody Technique; D007202:Indicators and Reagents; D051379:Mice; D008807:Mice, Inbred BALB C; D011506:Proteins / Q000302:isolation & purification*; D016271:Proto-Oncogene Proteins c-myc / Q000276:immunology* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007202:Indicators and Reagents; D011506:Proteins; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins