期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 98, 期 12, 页码 6554-6559出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.121178598
关键词
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资金
- NIGMS NIH HHS [GM190402] Funding Source: Medline
We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue. [SeMet(7)]cyt b562. by thioester-mediated chemical ligation of unprotected peptide segments. A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used. A cleavable thiol-containing auxiliary group. 1-phenyl-2-mercaptoethyl. was added to the alpha -amino group of one peptide segment to facilitate amide bond-forming ligation. The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis. Following native chemical ligation with a thioester-containing segment. the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a full-length unmodified polypeptide product. The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule. Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered [SeMet(7)]cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by approximate to 45 mV. The use of the 1-phenyl-2-mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments.
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