Naslov
Partial sequencing and immunochemical characterization of recombinant human cytokine

Autori
Knežević, Natalija ; Čepo, Tina ; Cindrić, Mario ; Kozlović, Marija ; Mildner, Boris

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Congress of the Croatian Society of Biochemistry and Molecular Biology with international participation, HDBMB 2004, Book of Abstracts / Dumić, Jerka - Zagreb : Farmaceutsko-biokemijski fakultet Sveučilišta u Zagrebu, 2004, 152-152

Skup
Congress of the Croatian Society of Biochemistry and Molecular Biology, HDBMB 2004

Mjesto i datum
HOC Bjelolasica, Hrvatska, 30.09.2004. - 02.10.2004

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
sequencing; human cytokine; immunchemical characterization

Sažetak
Recombinant human cytokine (18, 800 Da) is a monomeric protein that contains 175 amino acids and two disulphide bonds. Here we report confirmation of its primary structure, partial sequencing 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 Proteinlynx software (Micromass, UK) we predicted amino acid sequence and molecular masses of peptides derived after cleavage with endoproteinase Glu-C (Staphylococcus aureus V 8, Roche) and trypsin (from porcine pancreas, Merck). Endoproteinase Glu-C cleaves peptide bonds C-terminally at glutamic and aspartic acid, whereas trypsin is used for cleaving peptide bonds after lysine and arginine (if not followed by proline). After digestion with endoproteinase Glu-C we identified 10 of 12 predicted fragments. After digestion with trypsin 7 of 10 predicted fragments were identified. Derived fragments were separated by reverse phase liquid chromatography. Peptides were identified by mass spectrometry. Using this method 95% of protein sequence was confirmed. Sequence of four fragments was further confirmed by LC-MS/MS. Separated fragments were isolated in order to define epitope regions. Peptides generated using described methods of preparation were denatured and reduced. Each fragment was further characterized by immunochemical reaction with specific monoclonal antibodies using DOT-BLOT method.

Izvorni jezik
Engleski

Znanstvena područja
Farmacija, Biotehnologija

Napomena
Rad je izrađen u PLIVA Istraživanje i Razvoj d.o.o. i PLIVA Istraživački Institut d.o.o. Rad nije financiran od Ministarstva znanosti, obrazovanja i športa RH.

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