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Selective reduction...
Selective reduction and chemical modification of oxidized lipase cysteine mutants
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- Hedin, Eva. M. K. (författare)
- KTH,Bioteknologi
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- Patkar, S. A. (författare)
- KTH,Bioteknologi
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- Vind, J. (författare)
- KTH,Bioteknologi
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- Svendsen, A. (författare)
- KTH,Bioteknologi
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- Hult, Karl (författare)
- KTH,Biokemi och biokemisk teknologi
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- Berglund, Per (författare)
- KTH,Biokemi och biokemisk teknologi
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(creator_code:org_t)
- Canadian Science Publishing, 2002
- 2002
- Engelska.
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Ingår i: Canadian journal of chemistry (Print). - : Canadian Science Publishing. - 0008-4042 .- 1480-3291. ; 80:6, s. 529-539
- Relaterad länk:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- 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.
Ämnesord
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinsk bioteknologi -- Medicinsk bioteknologi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Medical Biotechnology -- Medical Biotechnology (hsv//eng)
Nyckelord
- lipase
- cysteine mutant
- selective reduction
- chemical modification
- methanethiosulfonate
- humicola-lanuginosa lipase
- fluorescence spectroscopy
- conformational-changes
- protein
- stability
- sulfhydryl
- dynamics
- enzymes
- binding
- thiols
- Biochemistry
- Biokemi
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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