Engineering unusual amino acids into peptides using lantibiotic synthetase.
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.
Section of Infection Biology, Department of Functional Bioscience, Fukuoka Dental College, Fukuoka 814-0913, Japan. jungao@college.fdcnet.ac.jp
Jun-Ichi
J
Nagao
Kouki
K
Shioya
Yoshitaka
Y
Harada
Ken-Ichi
K
Okuda
Takeshi
T
Zendo
Jiro
J
Nakayama
Kenji
K
Sonomoto
D000596:Amino Acids; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006836:Hydro-Lyases / Q000378:metabolism*; D010453:Peptide Synthases / Q000378:metabolism*; D010455:Peptides; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis*
D000596:Amino Acids; D029968:Escherichia coli Proteins; D010455:Peptides; D011994:Recombinant Proteins; D006836:Hydro-Lyases; C505225:NukM protein, E coli; D010453:Peptide Synthases