TY - JOUR
T1 - Solid‐phase synthesis of bovine pancreatic trypsin inhibitor BPTI) and two analogues
T2 - A chemical approach for evaluating the role of disulfide bridges in protein folding and stability
AU - FERRER, MARC
AU - WOODWARD, CLARE
AU - Barany, George
N1 - Copyright:
Copyright 2016 Elsevier B.V., All rights reserved.
PY - 1992/9
Y1 - 1992/9
N2 - 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 α‐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 sup‐port, 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.
AB - 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 α‐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 sup‐port, 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.
KW - Fmoc solid‐phase peptide synthesis
KW - and stability
KW - bovine pancreatic trypsin inhibitor (BPTI)
KW - conformation
KW - disulfide bridges
KW - half‐cystine
KW - protein folding
KW - α‐amino‐n‐butyric acid
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U2 - 10.1111/j.1399-3011.1992.tb00292.x
DO - 10.1111/j.1399-3011.1992.tb00292.x
M3 - Article
C2 - 1282503
AN - SCOPUS:0026483350
SN - 0367-8377
VL - 40
SP - 194
EP - 207
JO - International Journal of Peptide and Protein Research
JF - International Journal of Peptide and Protein Research
IS - 3-4
ER -