Search: id:"swepub:oai:DiVA.org:kth-21725" >
Selective reduction...
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Hedin, Eva. M. K.KTH,Bioteknologi
(author)
Selective reduction and chemical modification of oxidized lipase cysteine mutants
- Article/chapterEnglish2002
Publisher, publication year, extent ...
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Canadian Science Publishing,2002
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printrdacarrier
Numbers
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LIBRIS-ID:oai:DiVA.org:kth-21725
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https://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-21725URI
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https://doi.org/10.1139/v02-046DOI
Supplementary language notes
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Language:English
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Summary in:English
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Subject category:ref swepub-contenttype
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Subject category:art swepub-publicationtype
Notes
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QC 20100525
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Thirteen single-cysteine mutants of the 33 kDa fungal triacylglycerol lipase Thermomyces (formerly Humicola) lanuginosa lipase (TLL, EC 3.1.1.3) Were produced and characterized for the purpose of site-directed chemical modification with spectroscopic reporter groups. All cysteine mutants were found to be predominantly blocked by oxidation to disulfides with endogenous cysteine during production. The fraction of lipase molecules with free sulfhydryl groups was analyzed by labeling with N-biotinylaminoethyl methanethiosulfonate, followed by a novel dot-blot method based on biotin-streptavidin interactions. A non-invasive method for the reduction of the introduced cysteine was elaborated for this protein containing three native disulfide bridges. The site-specifically reduced TLL mutants were then labeled with the sulfhydryl-specific reagents 2-(5-dimethylaminonaphth-1-ylsulfonamido)ethyl methanethiosulfonate or (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate, and studied by fluorescence and electron spin resonance (ESR) spectroscopy.
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Patkar, S. A.KTH,Bioteknologi
(author)
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Vind, J.KTH,Bioteknologi
(author)
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Svendsen, A.KTH,Bioteknologi
(author)
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Hult, KarlKTH,Biokemi och biokemisk teknologi(Swepub:kth)u1nzlwuj
(author)
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Berglund, PerKTH,Biokemi och biokemisk teknologi(Swepub:kth)u1271chh
(author)
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KTHBioteknologi
(creator_code:org_t)
Related titles
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In:Canadian journal of chemistry (Print): Canadian Science Publishing80:6, s. 529-5390008-40421480-3291
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