کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020712 1069200 2013 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Purification and use of E. coli peptide deformylase for peptide deprotection in chemoenzymatic peptide synthesis
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Purification and use of E. coli peptide deformylase for peptide deprotection in chemoenzymatic peptide synthesis
چکیده انگلیسی

Peptide deformylases (PDFs) catalyze the removal of the formyl group from the N-terminal methionine residue in nascent polypeptide chains in prokaryotes. Its deformylation activity makes PDF an attractive candidate for the biocatalytic deprotection of formylated peptides that are used in chemoenzymatic peptide synthesis. For this application it is essential to use PDF preparations that are free of contamination by peptidases that can cleave internal peptide bonds. Therefore, different purification methods were attempted and an industrially applicable purification procedure was developed based on a single anion-exchange chromatography step of an engineered PDF variant that was equipped with an anionic octaglutamate tag. The deformylation activity and stability of the engineered enzyme were similar to those of the wild-type PDF. This purification method furnished a PDF preparation with a 1500-fold decreased level of contamination by amidases and peptidases as compared to cell-free extract. It was shown that the enzyme could be used for deprotection of a formylated dipeptide that was prepared by thermolysin-mediated coupling.


► A derivative of E. coli peptide defomylase carrying an anionic octaglutamate tag was constructed by genetic engineering.
► The modified enzyme could be separated from host proteases by a cheap and simple anion-chromatography step.
► The modified peptide deformylase was used for deprotection of an enzymatically synthesized N-formyl-protected dipeptide.
► The peptide-deformylase treated dipeptide was used as a nucleophile in thermolysin-mediated synthesis of a tripeptide.
► The results show that peptide defomylase is an attractive catalyst for deprotection of N-formylated peptides.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 89, Issue 1, May 2013, Pages 73–79
نویسندگان
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