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D. V. Smirnova, M. I. Koksharov, I. N. Zorov, N. N. Ugarova
Fusion proteins streptavidin-luciferase.preparation
and properties
Abstract
Using
genetic engineering methods we constructed four plasmids encoding fusion
proteins of thermostable mutant (TS) Luciola mingrelica firefly
luciferase (luc) and streptavidin (SA) with His6 sequence on the N-
or C-terminus of fusions: SA-Luc-His6, His6-SA-Luc, Luc-SA-His6.The
fusion proteins were expressed and isolated via metal-chelate
chromatography. We studied their oligomeric
composition, bioluminescent activity, thermostability,
spectra of bioluminescence and biotin-binding ability. It was shown that the
total bioluminescent activity of fusion Luc-SA-His6 is 15%, and
bioluminescent activity for other proteins is in range 35–50 % from activity of
free luciferase TS. It was shown, that addition of SA
domain has no influence on bioluminescent spectra of proteins, but lead to two
time reduction of thermostability at 47°C. Size-exclusion chromatography showed that
according to plasmid structure, fusions form different oligomeric
compositions: dimeric, tetrameric
and other high molecular weight aggregates, which distinguish by their enzyme
activity and affinity to biotin. Fusion His6-SA-Luc has the best
properties.
Key words: firefly luciferase, Luciolamingrelica, fusion protein, streptavidin,
oligomeric structure
Copyright (C) Chemistry Dept., Moscow State University, 2002
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