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Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen

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Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen. / Rawlings, Andrea E.; Levdikov, Vladimir M.; Blagova, Elena; Colledge, Vicki L.; Mas, Philippe J.; Tunaley, James; Vavrova, Ludmila; Wilson, Keith S.; Barak, Imrich; Hart, Darren J.; Wilkinson, Anthony J.

In: PROTEIN ENGINEERING DESIGN, Vol. 23, No. 11, 11.2010, p. 817-825.

Research output: Contribution to journalArticlepeer-review

Harvard

Rawlings, AE, Levdikov, VM, Blagova, E, Colledge, VL, Mas, PJ, Tunaley, J, Vavrova, L, Wilson, KS, Barak, I, Hart, DJ & Wilkinson, AJ 2010, 'Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen', PROTEIN ENGINEERING DESIGN, vol. 23, no. 11, pp. 817-825. https://doi.org/10.1093/protein/gzq057

APA

Rawlings, A. E., Levdikov, V. M., Blagova, E., Colledge, V. L., Mas, P. J., Tunaley, J., Vavrova, L., Wilson, K. S., Barak, I., Hart, D. J., & Wilkinson, A. J. (2010). Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen. PROTEIN ENGINEERING DESIGN, 23(11), 817-825. https://doi.org/10.1093/protein/gzq057

Vancouver

Rawlings AE, Levdikov VM, Blagova E, Colledge VL, Mas PJ, Tunaley J et al. Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen. PROTEIN ENGINEERING DESIGN. 2010 Nov;23(11):817-825. https://doi.org/10.1093/protein/gzq057

Author

Rawlings, Andrea E. ; Levdikov, Vladimir M. ; Blagova, Elena ; Colledge, Vicki L. ; Mas, Philippe J. ; Tunaley, James ; Vavrova, Ludmila ; Wilson, Keith S. ; Barak, Imrich ; Hart, Darren J. ; Wilkinson, Anthony J. / Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen. In: PROTEIN ENGINEERING DESIGN. 2010 ; Vol. 23, No. 11. pp. 817-825.

Bibtex - Download

@article{ee960d8dd6074a0db0563caa9e51691a,
title = "Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen",
abstract = "SpoIIE is a dual function protein that plays important roles during sporulation in Bacillus subtilis. It binds to the tubulin-like protein FtsZ causing the cell division septum to relocate from mid-cell to the cell pole, and it dephosphorylates SpoIIAA phosphate leading to establishment of differential gene expression in the two compartments following the asymmetric septation. Its 872 residue polypeptide contains a multiple-membrane spanning sequence at the N-terminus and a PP2C phosphatase domain at the C-terminus. The central segment that binds to FtsZ is unlike domains of known structure or function, moreover the domain boundaries are poorly defined and this has hampered the expression of soluble fragments of SpoIIE at the levels required for structural studies. Here we have screened over 9000 genetic constructs of spoIIE using a random incremental truncation library approach, ESPRIT, to identify a number of soluble C-terminal fragments of SpoIIE that were aligned with the protein sequence to map putative domains and domain boundaries. The expression and purification of three fragments were optimised, yielding multimilligram quantities of the PP2C phosphatase domain, the putative FtsZ-binding domain and a larger fragment encompassing both these domains. All three fragments are monomeric and the PP2C domain-containing fragments have phosphatase activity.",
keywords = "SpoIIE, sporulation, ESPRIT, phosphatase, cell division, ANTI-SIGMA FACTOR, CELL-SPECIFIC TRANSCRIPTION, ASYMMETRIC DIVISION, SPORULATION SEPTUM, CRYSTAL-STRUCTURE, GENE-EXPRESSION, FTSZ, PHOSPHATASE, ESTABLISHMENT, LOCALIZATION",
author = "Rawlings, {Andrea E.} and Levdikov, {Vladimir M.} and Elena Blagova and Colledge, {Vicki L.} and Mas, {Philippe J.} and James Tunaley and Ludmila Vavrova and Wilson, {Keith S.} and Imrich Barak and Hart, {Darren J.} and Wilkinson, {Anthony J.}",
year = "2010",
month = nov,
doi = "10.1093/protein/gzq057",
language = "English",
volume = "23",
pages = "817--825",
journal = "PROTEIN ENGINEERING DESIGN",
issn = "1741-0126",
publisher = "Oxford University Press",
number = "11",

}

RIS (suitable for import to EndNote) - Download

TY - JOUR

T1 - Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen

AU - Rawlings, Andrea E.

AU - Levdikov, Vladimir M.

AU - Blagova, Elena

AU - Colledge, Vicki L.

AU - Mas, Philippe J.

AU - Tunaley, James

AU - Vavrova, Ludmila

AU - Wilson, Keith S.

AU - Barak, Imrich

AU - Hart, Darren J.

AU - Wilkinson, Anthony J.

PY - 2010/11

Y1 - 2010/11

N2 - SpoIIE is a dual function protein that plays important roles during sporulation in Bacillus subtilis. It binds to the tubulin-like protein FtsZ causing the cell division septum to relocate from mid-cell to the cell pole, and it dephosphorylates SpoIIAA phosphate leading to establishment of differential gene expression in the two compartments following the asymmetric septation. Its 872 residue polypeptide contains a multiple-membrane spanning sequence at the N-terminus and a PP2C phosphatase domain at the C-terminus. The central segment that binds to FtsZ is unlike domains of known structure or function, moreover the domain boundaries are poorly defined and this has hampered the expression of soluble fragments of SpoIIE at the levels required for structural studies. Here we have screened over 9000 genetic constructs of spoIIE using a random incremental truncation library approach, ESPRIT, to identify a number of soluble C-terminal fragments of SpoIIE that were aligned with the protein sequence to map putative domains and domain boundaries. The expression and purification of three fragments were optimised, yielding multimilligram quantities of the PP2C phosphatase domain, the putative FtsZ-binding domain and a larger fragment encompassing both these domains. All three fragments are monomeric and the PP2C domain-containing fragments have phosphatase activity.

AB - SpoIIE is a dual function protein that plays important roles during sporulation in Bacillus subtilis. It binds to the tubulin-like protein FtsZ causing the cell division septum to relocate from mid-cell to the cell pole, and it dephosphorylates SpoIIAA phosphate leading to establishment of differential gene expression in the two compartments following the asymmetric septation. Its 872 residue polypeptide contains a multiple-membrane spanning sequence at the N-terminus and a PP2C phosphatase domain at the C-terminus. The central segment that binds to FtsZ is unlike domains of known structure or function, moreover the domain boundaries are poorly defined and this has hampered the expression of soluble fragments of SpoIIE at the levels required for structural studies. Here we have screened over 9000 genetic constructs of spoIIE using a random incremental truncation library approach, ESPRIT, to identify a number of soluble C-terminal fragments of SpoIIE that were aligned with the protein sequence to map putative domains and domain boundaries. The expression and purification of three fragments were optimised, yielding multimilligram quantities of the PP2C phosphatase domain, the putative FtsZ-binding domain and a larger fragment encompassing both these domains. All three fragments are monomeric and the PP2C domain-containing fragments have phosphatase activity.

KW - SpoIIE

KW - sporulation

KW - ESPRIT

KW - phosphatase

KW - cell division

KW - ANTI-SIGMA FACTOR

KW - CELL-SPECIFIC TRANSCRIPTION

KW - ASYMMETRIC DIVISION

KW - SPORULATION SEPTUM

KW - CRYSTAL-STRUCTURE

KW - GENE-EXPRESSION

KW - FTSZ

KW - PHOSPHATASE

KW - ESTABLISHMENT

KW - LOCALIZATION

UR - http://www.scopus.com/inward/record.url?scp=77958163395&partnerID=8YFLogxK

U2 - 10.1093/protein/gzq057

DO - 10.1093/protein/gzq057

M3 - Article

VL - 23

SP - 817

EP - 825

JO - PROTEIN ENGINEERING DESIGN

JF - PROTEIN ENGINEERING DESIGN

SN - 1741-0126

IS - 11

ER -