Multifunctional g3p-peptide tag for current phage display systems.
We have previously described a monoclonal antibody (mAb), 10C3, directed against the gene-3 protein (g3p) of filamentous phage M13, which was produced to study g3p fusion protein expression in Escherichia coli and its incorporation in the phage capsid [Tesar, M., Beckmann, C., Röttgen, P., Haase, B., Faude, U., Timmis, K., 1995. Monoclonal antibody against pIII of filamentous phage: an immunological tool to study pIII fusion protein expression in phage display systems. Immunology 1, 53-54]. In this study we report mapping of the antigenic epitope of the mAb 10C3, by means of short overlapping peptide-sequences [Frank, R., Overwin, H., 1996. Spot synthesis. In: Morris, G.E. (Ed.), Methods in Molecular Biology, Vol. 66: Epitope Mapping Protocols. Humana Press, Totowa, NJ, pp. 149-169.] comprising the C-terminal half of the g3-protein. A minimal recognizable peptide was found which is represented in the 11 amino acid sequence from positions 292 to 302 of g3p [Wezenbeek van, P.M.G.P., Hulsebos, T.J.M., Schoenmakers, J.G.G., 1980. Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd. Gene 11, 129-148]. In order to use the antibody also for detection and purification of recombinant proteins, such as single chain antibodies, the epitope was introduced as a tag sequence into the phagemid pHEN1 [Hoogenboom, H.R., Griffith, A.D., Johnson, K., Chiswell, D.J., Hudson, P., Winter, G., 1991. Multi-subunit proteins on the surface of the filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acid Res. 19, 4133-4137; Nissim, A., Hoogenboom, H.R., Tomlinson, I.M., Flynn, G., Midgley, C., Lane, D., Winter, G., 1994. Antibody fragments from a single pot phage display library as immunochemical reagents. EMBO J. 13 (3) 692-698]. Purified single chain antibodies containing this tag were detectable down to a concentration of 2 ng ml(-1) under non-denaturing conditions (ELISA) or 4 ng per lane on immunoblots. The high sensitivity of the antibody for the peptide tag was reflected in the antibody affinity constant K(D) of 6.80 x 10(-10) M, which was determined by real time biomolecular interaction analysis (BIA) based on surface plasmon resonance (SPR) [Karlsson, R., Fält, A., 1997. Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors. J. Immunol. Methods 200, 121-133]. Finally, recombinant proteins in E. coli periplasmic extracts could be purified in a single step by affinity purification using immobilized mAb 10C3. These studies demonstrated that the new peptide-tag and its corresponding mAb represents a versatile tool for the detection of recombinant proteins selected by phage display technology.
Beckmann
C
C
Division of Microbiology, GBF-National Research Centre for Biotechnology, Braunschweig, Germany.
Haase
B
B
Timmis
K N
KN
Tesar
M
M
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D017104:Bacteriophage M13 / Q000235:genetics / Q000276:immunology*; D015415:Biomarkers; D015374:Biosensing Techniques; D036022:Capsid Proteins; D003001:Cloning, Molecular / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology; D012680:Sensitivity and Specificity; D014760:Viral Fusion Proteins / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D015415:Biomarkers; D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D007128:Immunoglobulin Fragments; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; C055966:immunoglobulin Fv