Ascione_2012_Biochem.Biophys.Res.Commun_420_542

Reference

Title : Native expression and purification of hormone-sensitive lipase from Psychrobacter sp. TA144 enhances protein stability and activity - Ascione_2012_Biochem.Biophys.Res.Commun_420_542
Author(s) : Ascione G , de Pascale D , De Santi C , Pedone C , Dathan NA , Monti SM
Ref : Biochemical & Biophysical Research Communications , 420 :542 , 2012
Abstract :

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.

PubMedSearch : Ascione_2012_Biochem.Biophys.Res.Commun_420_542
PubMedID: 22440394
Gene_locus related to this paper: morsp-2lipa

Related information

Gene_locus morsp-2lipa

Citations formats

Ascione G, de Pascale D, De Santi C, Pedone C, Dathan NA, Monti SM (2012)
Native expression and purification of hormone-sensitive lipase from Psychrobacter sp. TA144 enhances protein stability and activity
Biochemical & Biophysical Research Communications 420 :542

Ascione G, de Pascale D, De Santi C, Pedone C, Dathan NA, Monti SM (2012)
Biochemical & Biophysical Research Communications 420 :542