Heterologous expression and purification of SpaB involved in subtilin biosynthesis

Lili Xie; Champak Chatterjee; Rashna Balsara; Nicole M. Okeley; Wilfred A. Van der Donk

doi:10.1016/S0006-291X(02)00783-0

Heterologous expression and purification of SpaB involved in subtilin biosynthesis

Lili Xie, Champak Chatterjee, Rashna Balsara, Nicole M. Okeley, Wilfred A. Van der Donk

Research output: Contribution to journal › Article › peer-review

Abstract

Lantibiotic peptides contain thioether bridges termed lanthionines that are putatively generated by dehydration of Ser and Thr residues followed by Michael addition of cysteine residues within the peptide. The LanB and LanC proteins have been proposed to catalyze the dehydration and formation of the thioether rings, respectively. We report here the first heterologous overexpression in Escherichia coli of SpaB, the putative dehydratase for subtilin. Sequence analysis of spaB revealed several nucleotide differences with current gene database entries. The solubility of SpaB was increased dramatically when co-expressed with GroEL/ES, and soluble His6-tagged SpaB was purified. The protein is at least a dimer, and interaction between SpaB and SpaC was observed. SpaS the putative substrate for SpaB was overexpressed in E. coli as an intein fusion protein, and after cleavage, the peptide was obtained in good yield.

Original language	English (US)
Pages (from-to)	952-957
Number of pages	6
Journal	Biochemical and Biophysical Research Communications
Volume	295
Issue number	4
DOIs	https://doi.org/10.1016/S0006-291X(02)00783-0
State	Published - 2002

Keywords

Dehydratase
Expressed protein ligation
Lantibiotics
Subtilin prepeptide

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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10.1016/S0006-291X(02)00783-0

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title = "Heterologous expression and purification of SpaB involved in subtilin biosynthesis",

abstract = "Lantibiotic peptides contain thioether bridges termed lanthionines that are putatively generated by dehydration of Ser and Thr residues followed by Michael addition of cysteine residues within the peptide. The LanB and LanC proteins have been proposed to catalyze the dehydration and formation of the thioether rings, respectively. We report here the first heterologous overexpression in Escherichia coli of SpaB, the putative dehydratase for subtilin. Sequence analysis of spaB revealed several nucleotide differences with current gene database entries. The solubility of SpaB was increased dramatically when co-expressed with GroEL/ES, and soluble His6-tagged SpaB was purified. The protein is at least a dimer, and interaction between SpaB and SpaC was observed. SpaS the putative substrate for SpaB was overexpressed in E. coli as an intein fusion protein, and after cleavage, the peptide was obtained in good yield.",

keywords = "Dehydratase, Expressed protein ligation, Lantibiotics, Subtilin prepeptide",

author = "Lili Xie and Champak Chatterjee and Rashna Balsara and Okeley, {Nicole M.} and {Van der Donk}, {Wilfred A.}",

note = "Funding Information: This work was supported by the National Institutes of Health (GM58822) and a Beckman Young Investigator Award. L.X. acknowledges support from Zeneca and Fuson Fellowships, and N.M.O. was supported in part by an Abbott Graduate Fellowship. W.A.V. is a Cottrell Scholar of the Research Corporation.",

year = "2002",

doi = "10.1016/S0006-291X(02)00783-0",

language = "English (US)",

volume = "295",

pages = "952--957",

journal = "Biochemical and Biophysical Research Communications",

issn = "0006-291X",

publisher = "Elsevier B.V.",

number = "4",

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T1 - Heterologous expression and purification of SpaB involved in subtilin biosynthesis

AU - Xie, Lili

AU - Chatterjee, Champak

AU - Balsara, Rashna

AU - Okeley, Nicole M.

AU - Van der Donk, Wilfred A.

N1 - Funding Information: This work was supported by the National Institutes of Health (GM58822) and a Beckman Young Investigator Award. L.X. acknowledges support from Zeneca and Fuson Fellowships, and N.M.O. was supported in part by an Abbott Graduate Fellowship. W.A.V. is a Cottrell Scholar of the Research Corporation.

PY - 2002

Y1 - 2002

N2 - Lantibiotic peptides contain thioether bridges termed lanthionines that are putatively generated by dehydration of Ser and Thr residues followed by Michael addition of cysteine residues within the peptide. The LanB and LanC proteins have been proposed to catalyze the dehydration and formation of the thioether rings, respectively. We report here the first heterologous overexpression in Escherichia coli of SpaB, the putative dehydratase for subtilin. Sequence analysis of spaB revealed several nucleotide differences with current gene database entries. The solubility of SpaB was increased dramatically when co-expressed with GroEL/ES, and soluble His6-tagged SpaB was purified. The protein is at least a dimer, and interaction between SpaB and SpaC was observed. SpaS the putative substrate for SpaB was overexpressed in E. coli as an intein fusion protein, and after cleavage, the peptide was obtained in good yield.

AB - Lantibiotic peptides contain thioether bridges termed lanthionines that are putatively generated by dehydration of Ser and Thr residues followed by Michael addition of cysteine residues within the peptide. The LanB and LanC proteins have been proposed to catalyze the dehydration and formation of the thioether rings, respectively. We report here the first heterologous overexpression in Escherichia coli of SpaB, the putative dehydratase for subtilin. Sequence analysis of spaB revealed several nucleotide differences with current gene database entries. The solubility of SpaB was increased dramatically when co-expressed with GroEL/ES, and soluble His6-tagged SpaB was purified. The protein is at least a dimer, and interaction between SpaB and SpaC was observed. SpaS the putative substrate for SpaB was overexpressed in E. coli as an intein fusion protein, and after cleavage, the peptide was obtained in good yield.

KW - Dehydratase

KW - Expressed protein ligation

KW - Lantibiotics

KW - Subtilin prepeptide

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JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

IS - 4

ER -

Heterologous expression and purification of SpaB involved in subtilin biosynthesis

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