Please use this identifier to cite or link to this item:
https://hdl.handle.net/2440/78426
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Type: | Journal article |
Title: | Cell-free protein synthesis of membrane (1,3)-beta-D-glucan (curdlan) synthase: Co-translational insertion in liposomes and reconstitution in nanodiscs |
Author: | Periasamy, A. Shadiac, N. Amalraj, A. Garajova, S. Nagarajan, Y. Waters, S. Mertens, H. Hrmova, M. |
Citation: | BBA: Biomembranes, 2013; 1828(2):743-757 |
Publisher: | Elsevier Science BV |
Issue Date: | 2013 |
ISSN: | 0005-2736 1879-2642 |
Statement of Responsibility: | Agalya Periasamy, Nadim Shadiac, Amritha Amalraj, Soňa Garajová, Yagnesh Nagarajan, Shane Waters, Haydyn D.T. Mertens, Maria Hrmova |
Abstract: | A membrane-embedded curdlan synthase (CrdS) from Agrobacterium is believed to catalyse a repetitive addition of glucosyl residues from UDP-glucose to produce the (1,3)-β-d-glucan (curdlan) polymer. We report wheat germ cell-free protein synthesis (WG-CFPS) of full-length CrdS containing a 6xHis affinity tag and either Factor Xa or Tobacco Etch Virus proteolytic sites, using a variety of hydrophobic membrane-mimicking environments. Full-length CrdS was synthesised with no variations in primary structure, following analysis of tryptic fragments by MALDI-TOF/TOF Mass Spectrometry. Preparative scale WG-CFPS in dialysis mode with Brij-58 yielded CrdS in mg/ml quantities. Analysis of structural and functional properties of CrdS during protein synthesis showed that CrdS was co-translationally inserted in DMPC liposomes during WG-CFPS, and these liposomes could be purified in a single step by density gradient floatation. Incorporated CrdS exhibited a random orientation topology. Following affinity purification of CrdS, the protein was reconstituted in nanodiscs with Escherichia coli lipids or POPC and a membrane scaffold protein MSP1E3D1. CrdS nanodiscs were characterised by small-angle X-ray scattering using synchrotron radiation and the data obtained were consistent with insertion of CrdS into bilayers. We found CrdS synthesised in the presence of the Ac-AAAAAAD surfactant peptide or co-translationally inserted in liposomes made from E. coli lipids to be catalytically competent. Conversely, CrdS synthesised with only Brij-58 was inactive. Our findings pave the way for future structural studies of this industrially important catalytic membrane protein. |
Keywords: | Family GT2 transferase Small-angle X-ray scattering Surfactant Surfactant peptide Topology (1,3)-β-d-Glucan |
Rights: | © 2012 Elsevier B.V. All rights reserved. |
DOI: | 10.1016/j.bbamem.2012.10.003 |
Grant ID: | http://purl.org/au-research/grants/arc/DP120100900 http://purl.org/au-research/grants/arc/LP120100201 http://purl.org/au-research/grants/arc/LP120100201 |
Published version: | http://dx.doi.org/10.1016/j.bbamem.2012.10.003 |
Appears in Collections: | Agriculture, Food and Wine publications Aurora harvest 4 |
Files in This Item:
File | Description | Size | Format | |
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hdl_78426.pdf | Accepted version | 10.31 MB | Adobe PDF | View/Open |
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