کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1944413 1053210 2013 15 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Cell-free protein synthesis of membrane (1,3)-β-d-glucan (curdlan) synthase: Co-translational insertion in liposomes and reconstitution in nanodiscs
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Cell-free protein synthesis of membrane (1,3)-β-d-glucan (curdlan) synthase: Co-translational insertion in liposomes and reconstitution in nanodiscs
چکیده انگلیسی

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.

Figure optionsDownload high-quality image (193 K)Download as PowerPoint slideHighlights
► Wheat germ cell-free protein synthesis affords a full-length curdlan synthase.
► Up to mg/ml yield of curdlan synthase was obtained in a dialysis mode.
► Active curdlan synthase was made with surfactant peptides or inserted in liposomes.
► Nanodiscs with curdlan synthase were characterised by small-angle X-ray scattering.
► Nanodiscs have a maximum at ~ 0.1 Å that corresponds to a phospholipid bilayer.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimica et Biophysica Acta (BBA) - Biomembranes - Volume 1828, Issue 2, February 2013, Pages 743–757
نویسندگان
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