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| SOLID-PHASE SYNTHESIS OF BOVINE PANCREATIC TRYPSIN-INHIBITOR (BPTI) AND 2 ANALOGS - A CHEMICAL APPROACH FOR EVALUATING THE ROLE OF DISULFIDE BRIDGES IN PROTEIN FOLDING AND STABILITY |
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| Author(s): FERRER M, WOODWARD C, BARANY G |
| Source: INTERNATIONAL JOURNAL OF PEPTIDE AND PROTEIN RESEARCH Volume: 40 Issue: 3-4 Pages: 194-207 Published: SEP-OCT 1992 |
| Times Cited: 46 References: 70 |
| Abstract: The linear sequence of bovine pancreatic trypsin inhibitor (BPTI) has been assembled by stepwise Fmoc solid-phase peptide synthesis on a polyethylene glycol-polystyrene (PEG-PS) graft support with p-alkoxybenzyl ester anchoring. Similar methods were used to prepare two analogues, the first with all six half-cystine (Cys) residues replaced by alpha-amino-n-butyric acid (Abu), and the second with replacement of Abu at four Cys positions while retaining the native pairing between positions 14 and 38. Following cleavage from the support, the linear molecules (reduced form) were purified by semipreparative reversed-phase high performance liquid chromatography (HPLC). The native structure of BPTI was then formed by oxidation of a dilute solution of the protein at pH 8.7 in the presence of oxidized glutathione. The BPTI analogue with one disulfide bridge was obtained following treatment with dimethyl sulfoxide (DMSO)-pH 6 buffer (1:9). Overall yields of homogeneous proteins were 2-4%, and further characterization was provided by amino acid analysis, sequencing, ion electrospray mass spectrometry, analytical HPLC, and capillary zone electrophoresis (CZE). Purified synthetic BPTI with the native sequence was indistinguishable from natural material by the analytical and biophysical criteria applied, including circular dichroism (CD) spectra and inhibition of trypsin action. Studies are in progress to evaluate conformational features of the analogues which respectively lack two, or all three, of the native disulfide bridges. |
| Document Type: Article |
| Language: English |
Addresses:
1. UNIV MINNESOTA, DEPT CHEM, 207 PLEASANT ST SE, MINNEAPOLIS, MN 55455 USA
2. UNIV MINNESOTA, DEPT BIOCHEM, ST PAUL, MN 55108 USA |
| Publisher: MUNKSGAARD INT PUBL LTD, 35 NORRE SOGADE, PO BOX 2148, DK-1016 COPENHAGEN, DENMARK |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: JX365 |
| ISSN: 0367-8377 |
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