ORCID Profile
0000-0001-8032-9700
Current Organisations
University of Leeds
,
University of Helsinki
,
Nankai University
,
BioCity Turku
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Publisher: Elsevier BV
Date: 09-2018
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 02-2007
Publisher: Oxford University Press (OUP)
Date: 03-2019
Abstract: In the accompanying paper, we described evolving a lipase to the point where variants were soluble, stable and capable of degrading C8 TAG and C8 esters. These variants were tested for their ability to survive in an environment that might be encountered in a washing machine. Unfortunately, they were inactivated both by treatment with a protease used in laundry detergents and by very low concentrations of sodium dodecyl sulfate (SDS). In addition, all the variants had very low levels of activity with triglycerides with long aliphatic chains and with naturally occurring oils, like olive oil. Directed evolution was used to select variants with enhanced properties. In the first 10 rounds of evolution, the primary screen was selected for variants capable of hydrolyzing olive oil whereas the secondary screen was selected for enhanced tolerance towards a protease and SDS. In the final six rounds of evolution, the primary and secondary screens identified variants that retained activity after treatment with SDS. Sixteen cycles of evolution gave variants with greatly enhanced lipolytic activity on substrates that had both long (C16 and C18) as well as short (C3 and C8) chains. We found variants that were stable for more than 3 hours in protease concentrations that rapidly degrade the wild-type enzyme. Enhanced tolerance towards SDS was found in variants that could break down naturally occurring lipid and resist protease attack. The amino acid changes that gave enhanced properties were concentrated in the cap domain responsible for substrate binding.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 10-07-2012
Abstract: Many laboratories use recombinant UDP-glucuronosyltransferases (UGTs), expressed in baculovirus-infected insect cells, for drug glucuronidation studies. We have infected Sf9 insect cells with increasing amounts of recombinant baculovirus, encoding either UGT1A9 or UGT2B7, and measured both glucuronidation activity and immunodetectable UGT in the resulting cell homogenates. The correlation between glucuronidation rates and degree of infection followed different trends, depending on whether activity was the actual activity measured or was corrected for UGT expression level. Above a certain low level of infection, further increases in infection ratios led to a large decline in normalized activity, presumably due to the presence of full-length but inactive enzyme in the s le. Because immunodetection does not distinguish between active and inactive UGT, comparison of normalized activity between different batches of a recombinant UGT, mutants of a given UGT, or different UGTs is prone to large inaccuracies. Such inaccuracies could be reduced by lowering the degree of infection of the insect cells, in combination with careful monitoring of UGT expression. However, the latter requires suitable antibodies for comparing UGT expression levels among preparations, antibodies that are not always available. Poly-His (His-tag)-containing peptides, fused to the UGT C terminus, allow sensitive immunodetection of expressed enzymes with monoclonal antibodies. We have now carefully examined the effects of two such fusion peptides on enzyme kinetics. A minor increase in the K(m) values has been detected in the His-tagged UGTs, but no changes in parameters such as the kinetic model and the effects of albumin addition.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Adrian Goldman.