Native expression and purification of hormone-sensitive lipase from Psychrobacter sp. TA144 enhances protein stability and activity(665 views) Ascione G, de Pascale D, De Santi C, Pedone C, Dathan NA, Monti SM
Institute of Biostructures and Bioimaging, National Research Council, Via Mezzocannone 16, I-80134 Naples, Italy.
Institute of Protein Biochemistry, National Research Council, Via Pietro Castellino 111, I-80131 Naples, Italy
References: Not available.
Native expression and purification of hormone-sensitive lipase from Psychrobacter sp. TA144 enhances protein stability and activity
Psychrobacter, a micro-organism originally isolated from Antarctic sea water, expresses an extremely active hormone-sensitive lipase (HSL) which catalyzes the hydrolysis of fatty acid esters at very low temperature and is therefore of great potential industrial and pharmaceutical interest. An insoluble form of the entire enzyme has previously been cloned and expressed in Escherichia coli, subsequently refolded and shown to be active, whilst a shorter but completely inactive version, lacking the N-terminal 98 amino acids has been expressed in soluble form. In this study the entire enzyme has been expressed as a fully soluble protein in E. coli in the presence of either the osmolyte trehalose, plus high salt concentration, or the membrane fluidizer benzyl alcohol. Trehalose promotes protein mono-dispersion by increasing the viscosity of the growth medium for bacterial cells, thereby helping circumvent protein aggregation, whilst the heat-shock inducer benzyl alcohol stimulates the production of a network of endogenous chaperones which actively prevent protein misfolding, whilst also converting recombinant aggregates to native, correctly folded proteins. The resultant recombinant protein proved to be more stable than its previously expressed counterpart, as shown by CD and enzymatic activity data which proved the enzyme to be more active at a higher temperature than its refolded counterpart. By light scattering analysis it was shown that the newly expressed protein was monomeric. The stability of the full length native protein will help in understanding the structure of PsyHSL and the role of its regulatory N-terminal for eventual application in a myriad of biotechnological processes. Copyright 2012 Elsevier Inc. All rights reserved.
Native expression and purification of hormone-sensitive lipase from Psychrobacter sp. TA144 enhances protein stability and activity
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Native expression and purification of hormone-sensitive lipase from Psychrobacter sp. TA144 enhances protein stability and activity
Aloj L, Aurilio M, Rinaldi V, D'Ambrosio L, Tesauro D, Peitl PK, Maina T, Mansi R, Von Guggenberg E, Joosten L, Sosabowski JK, Breeman WA, De Blois E, Koelewijn S, Melis M, Waser B, Beetschen K, Reubi JC, De Jong M * The EEE project(545 views) Proc Int Cosm Ray Conf Icrc Universidad Nacional Autonoma De Mexico, 2007; 5(HEPART2): 977-980. Impact Factor:0 ViewExport to BibTeXExport to EndNote