Lanthionine introduction into nukacin ISK-1 prepeptide by co-expression with modification enzyme NukM in Escherichia coli.
We demonstrated lanthionine introduction into hexa-histidine-tagged (His-tagged) nukacin ISK-1 prepeptide NukA by modification enzyme NukM in Escherichia coli. Co-expression of nukA and nukM, purification of the resulting His-tagged prepeptide by affinity chromatography, and subsequent mass spectrometry analysis showed that the prepeptide was converted into a postulated peptide with decrease in mass of 72Da which resulted from dehydration of four amino acids. Characterization of the resultant prepeptide indicated the presence of unusual amino acids, such as dehydrated amino acid, lanthionine or 3-methyllanthionine, in its C-terminal propeptide moiety. The modified prepeptide encompassing the leader peptide attached to the post-translationally modified propeptide moiety was readily obtained by one-step purification. Our findings will thus be a powerful tool for introducing unusual amino acids aimed at peptide engineering and also helpful to provide new insight for further understanding of lanthionine-forming enzymes for lantibiotics.
Nagao
Jun-Ichi
J
Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Harada
Yoshitaka
Y
Shioya
Kouki
K
Aso
Yuji
Y
Zendo
Takeshi
T
Nakayama
Jiro
J
Sonomoto
Kenji
K
D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001430:Bacteriocins / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013440:Sulfides / Q000737:chemistry* / Q000378:metabolism*
D001430:Bacteriocins; D029968:Escherichia coli Proteins; D010455:Peptides; D011994:Recombinant Proteins; D013440:Sulfides; C421186:nukacin ISK-1; D006836:Hydro-Lyases; C505225:NukM protein, E coli; C001520:lanthionine; D000409:Alanine