| Autor: |
Ken Ichi Okuda, Jun-ichi Nagao, Takeshi Zendo, Yoshitaka Harada, Kenji Sonomoto, Jiro Nakayama, Kouki Shioya |
| Rok vydání: |
2010 |
| Předmět: |
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| Zdroj: |
Methods in Molecular Biology ISBN: 9781617379666 |
| DOI: |
10.1007/978-1-61737-967-3_13 |
| Popis: |
Alteration of protein structure and function by introducing unusual amino acids has great potential to develop new biological tool and to produce novel therapeutic agents. Lantibiotics produced by Gram-positive bacteria are ribosomally synthesized and post-translationally modified antimicrobial peptides. The modification enzyme involved in lantibiotic biosynthesis can catalyze the formation of unusual amino acids in the nascent lantibiotic prepeptide. Here, a novel methodology on the lantibiotic nukacin ISK-1 is described for engineering unusual amino acid residues into hexa-histidine-tagged (His-tagged) prepeptide NukA by the modification enzyme NukM in Escherichia coli. Co-expression of His-tagged NukA and NukM, purification of the resulting His-tagged prepeptide by affinity chromatography, and subsequent mass spectrometry analysis show that the prepeptide is converted into a postulated peptide with decrease in mass which results from the formation of unusual amino acids such as dehydrated amino acid, lanthionine, or 3-methyl lanthionine at the expected positions. The modified prepeptide can be readily obtained by one-step purification. This strategy will thus be a simple and powerful tool for introducing unusual amino acid residues aimed at peptide engineering. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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