Expression and crystallization of a bacterial glycoside hydrolase family 116 β‐glucosidase from Thermoanaerobacterium xylanolyticum. Issue 1 (1st January 2015)

• Record Type: Journal Article
• Authors: Sansenya, Sompong; Mutoh, Risa; Charoenwattanasatien, Ratana; Kurisu, Genji; Ketudat Cairns, James R.
• Is Part Of: Acta crystallographica. Volume 71:Issue 1(2015:Jan.)
• Page Start: 41
• Page End: 44
• Publication Date: 2015-01-01
• Subjects: Crystallography -- Periodicals; Crystals -- Periodicals; 548
• DOI: 10.1107/S2053230X14025461
• Languages: English
• ISSNs: 2053-230X

Abstract

<abstract abstract-type="main" xml:lang="en"> <title> <x xml:space="preserve">Abstract</x> </title> <p>The <italic>Thermoanaerobacterium xylanolyticum</italic> gene product TxGH116, a glycoside hydrolase family 116 protein of 806 amino‐acid residues sharing 37% amino‐acid sequence identity over 783 residues with human glucosylceramidase 2 (GBA2), was expressed in <italic>Escherichia coli</italic>. Purification by heating, immobilized metal‐affinity and size‐exclusion chromatography produced &gt;90% pure TxGH116 protein with an apparent molecular mass of 90 kDa on SDS–PAGE. The purified TxGH116 enzyme hydrolyzed the <italic>p</italic>‐nitrophenyl (<italic>p</italic>NP) glycosides <italic>p</italic>NP‐β‐D‐glucoside, <italic>p</italic>NP‐β‐D‐galactoside and <italic>p</italic>NP‐<italic>N</italic>‐acetyl‐β‐D‐glucopyranoside, as well as cellobiose and cellotriose. The TxGH116 protein was crystallized using a precipitant consisting of 0.6 <italic>M</italic> sodium citrate tribasic, 0.1 <italic>M</italic> Tris–HCl pH 7.0 by vapour diffusion with micro‐seeding to form crystals with maximum dimensions of 120 × 25 × 5 µm. The TxGH116 crystals diffracted X‐rays to 3.15 Å resolution and belonged to the monoclinic space group <italic>P</italic>2₁. Structure solution will allow a structural explanation of the effects of human GBA2 mutations.</p> </abstract>