Cell-free protein synthesis of membrane (1,3)-¦Â-d-glucan (curdlan) synthase: Co-translational insertion in liposomes and reconstitution in nanodiscs

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• 作者：Agalya Periasamy^a ; ¹ ; Nadim Shadiac^a ; ¹ ; Amritha Amalraj^a ; So&#328 ; a Garajová ; ^a ; ² ; Yagnesh Nagarajan^a ; Shane Waters^a ; Haydyn D.T. Mertens^b ; Maria Hrmova^a ; ^{maria.hrmova@adelaide.edu.au}
• 关键词：¦Â-OG ; n-octyl-¦Â-d-glucopyranoside ; Brij-58 ; polyoxyethylene-(20)-cetyl-ether (C16&frasl ; 20) ; CAZy ; Carbohydrate-Active enZymes ; CF ; cell-free ; CFPS ; cell-free protein synthesis ; CHAPS ; 3-[(3-cholamidopropyl)dimethylammonio]-1-propansulfonate ; CM ; cataly
• 刊名：Biochimica et Biophysica Acta (BBA)/Biomembranes
• 出版年：2013
• 出版时间：February, 2013
• 年：2013
• 卷：1828
• 期：2
• 页码：743-757
• 全文大小：2101 K

文摘

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)-¦Â-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.