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Peptide mapping and epitope screening of recombinant human cytokine

Recombinant human cytokine of 18, 800 Da is a monomeric protein containing 175 amino acids and two disulphide bonds (1). Here we report primary structure confirmation and determination of regions recognized by specific antibodies using peptide mapping and epitope mapping methods. Peptide mapping is a method for confirmation and determination of amino acid sequence of proteins. The basic principle is protein cleveage with specific proteolytic enzymes or chemical reagents. Using BioLynx sofware (Micromass, UK) we predicted amino acid sequence and molecular masses of peptides derived after clevage with endoproteinase Glu-C (Staphylococcus aureus V 8) and trypsin (from porcine pancreas). Endoproteinase Glu-C cleaves peptide bonds C-terminally at aspartic acid, whereas trypsin is used for protein cleveage C-terminally at lysine and arginine (if not followed by proline)(2). After digestion with endoproteinase Glu-C and trypsin we identified 9 of 12 and 7 of 10 predicted fragments, respectively. Derived fragments were separated by reverse phase liquid chromatography and further identified by mass spectrometry method using single quadropole analyzer. Using this method 90% of protein sequence was confirmed. Separated fragments were isolated by fraction collector in order to define epitope regions. Protein regions recognized by specific antibody are called epitopes and are usually three-dimensional. Peptides generated using described methods are denaturated and reduced. Each fragment was characterized by immunochemical reaction with specific monoclonal antibodies using DOT-BLOT method (3, 4). Positive results were achieved for five fragments: A1 (1-20aa), A4 (35-47 aa), A7 (100-105 aa), A8 (106-110). We propose this method for screening residues involved in binding of specific antibodies. 1) H.S. Lu et al, Arch. Biochem. Biophys.: 268 (1989): 81-92 2) R. Perlman, J. Wang (Eds.), Formulation, Characterization, and Stability of Protein Drugs, Plenum Press, NY, 1996. 3)D. Voet, J. G. Voet, Biochemistry, J. Wiley and Sons, NY, 1996. 4) E. Nice et al:J. Chrom. 646(1993): 159-168.

recombinant human cytokine; peptide mapping; epitope mapping; protease

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