Quantitative analysis of c-myc-tagged protein in crude cell extracts using fluorescence polarization.
A fluorescence polarization (FP) assay was developed to determine the concentration of a c-myc-tagged recombinant protein in a crude cell extract. The basis of the assay was a competition between a c-myc-tagged protein and a fluorescein-labeled c-myc peptide for a c-myc antibody Fab. Fluorescein-labeled c-myc peptide produced a high-fluorescence polarization signal upon binding to the c-myc antibody, which can be inhibited in the presence of a c-myc-tagged protein. Quantitation of a c-myc-tagged protein was realized by measuring the decrease in fluorescence polarization. The observed IC(50) values in the competition FP assay were similar among all monomeric c-myc-tagged proteins tested, indicating that the interaction of the c-myc tag with the antibody was independent of the fusion protein sequence. The c-myc-tagged protein concentrations measured by FP were found to correlate well with values derived from a spike experiment and with values obtained by quantitative immunoblot. This assay was not perturbed by the presence of crude cell lysate, dithiothreitol or detergents, and worked with both native and denatured samples from several expression systems, including Escherichia coli, Pichia, insect cells, and mammalian cells. The assay under the current condition can detect as low as 0.05% expression level of c-myc-tagged protein with regards to total proteins, depending on the expression system. This assay is both quantitative and rapid (less than 15min) and is therefore suitable for the optimization of recombinant protein expression conditions as well as for the monitoring of protein purification procedures.
Applied Biotechnology Department, Bristol-Myers Squibb Pharma Company, Experimental Station, Wilmington, DE 19880-0336, USA. shaoxian.sun@pfizer.com
Shaoxian
S
Sun
Lien-Hanh T
LH
Nguyen
O
O
Harold Ross
Gregory F
GF
Hollis
Richard
R
Wynn
D001667:Binding, Competitive / Q000187:drug effects; D002457:Cell Extracts / Q000737:chemistry*; D002460:Cell Line; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli; D019793:Fluorescein / Q000032:analysis; D005454:Fluorescence Polarization / Q000379:methods*; D006801:Humans; D015151:Immunoblotting; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D010843:Pichia; D011489:Protein Denaturation; D016271:Proto-Oncogene Proteins c-myc / Q000032:analysis* / Q000276:immunology; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000276:immunology; D013997:Time Factors; D014157:Transcription Factors / Q000032:analysis
D002457:Cell Extracts; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D016271:Proto-Oncogene Proteins c-myc; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004229:Dithiothreitol; D019793:Fluorescein