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Identification and characterization of Fluorescent Protein from marine organisms and potentially applications

von Dr. Tiziana Masullo

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[1.] Tim/Fragment 010 01 - Diskussion
Zuletzt bearbeitet: 2014-11-27 19:50:21 Hindemith
BauernOpfer, Fragment, Gesichtet, SMWFragment, Schutzlevel sysop, Tim, Wiedenmann 2000

Typus
BauernOpfer
Bearbeiter
SleepyHollow02
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 10, Zeilen: 1 ff. (entire page)
Quelle: Wiedenmann 2000
Seite(n): 14092, 14093, Zeilen: 14092: r.col: last lines; 14093: l.col: 1ff
Molecular masses.

Wiedenmann et al. (2000) purified all proteins to near homogeneity. Both AsGFP 499/522 could not be separated by the purification steps. The molecular masses, determined by SDS-PAGE, are 26.2 kDA for the mixed fraction of AsGFP499/522 and 19.1 kDa for both asFP595 and asCP562. In gel filtration experiments under physiological conditions all protein pigments show apparent molecular masses of 66 kDa. For the GFPs this indicates a natural occurrence as dimers or trimers. Under denaturing conditions these oligomers can be partially split into monomers with apparent molecular masses of 23 kDa. Molecular masses for the AsGFP499 and asCP562 cloned were 25.4 kDa and 16.5 kDa.

Stability.

All colored proteins show a remarkable stability after treatment with heat, detergent, chaotrop, reducing agent, and pH extremes (Table 2. appendixes). Thermostability of asFP595 fluorescence is clearly increased compared with that of AsGFP499/522. In contrast, fluorescence of AsGFP499 is more stable when the solutions are exposed to 1% SDS, 8 M urea, and pH 11. Fluorescence of AsGFP499/522 and asFP595 is also stable when the proteins are treated with 4% paraformaldehyde (Wiedenmann et al., 2000).

Reversible denaturation.

Wiedenmann et al. (2000) heat-denatured the partially purified extracts of the proteins asFP595 and asCP562 in the presence of 2% SDS and 10% β-mercaptoethanol. After, proteins were separated by SDS/PAGE and transferred to a nitrocellulose membrane by semidry blotting. On the membrane, the band corresponding to asFP595 appears red under daylight conditions and exhibits orange fluorescence with an emission maximum at 595 nm under UV light. The fluorescence is stable and can even be detected after passage of the renatured protein through the membrane because of extended blotting. Renaturation with full restoration of fluorescence in the presence of β-mercaptoethanol has been described for heat-denatured GFP (Surpin & Ward, 1989). Surprisingly, asCP562, which is nonfluorescent in vivo, behaves in the same manner and shows identical fluorescence as renatured asFP595.

Molecular masses [...] All proteins were purified to near homogeneity. Both asFP499 and asFP522 could not be separated by the purification steps. The molecular masses, determined by

[page 14093]

SDS/PAGE are 26.2 kDA for the mixed fraction of asFP499/ asFP522 and 19.1 kDa for both asFP595 and asCP562 (Fig. 3 A and B). In gel filtration experiments under physiological conditions all protein pigments show apparent molecular masses of 66 kDa (Fig. 3.1 and 3.3). For the GFPs this indicates a natural occurrence as dimers or trimers (Fig. 3.3). Under denaturing conditions these oligomers can be partially split into monomers with apparent molecular masses of 23 kDa (Fig. 3.4). Stable dimers also are reported for GFP from Renilla and some other pennatularians (12, 27).

Stability. All colored proteins show a remarkable stability after treatment with heat, detergent, chaotrop, reducing agent, and pH extremes (Table 2). Thermostability of asFP595 fluorescence is clearly increased compared with that of asFP499/asFP522. In contrast, fluorescence of asFP499 is more stable when the solutions are exposed to 1% SDS, 8 M urea, and pH 11. Fluorescence of asFP499/asFP522 and asFP595 is also stable when the proteins are treated with 4% paraformaldehyde. Overall, the stability of the pigments from A. sulcata is comparable to that of the GFPs from Aequorea and Renilla (27–30).

Reversible denaturation. Partially purified extracts of the proteins asFP595 and asCP562 were heat-denatured in the presence of 2% SDS and 10% β-mercaptoethanol. Proteins were separated by SDS/PAGE and transferred to a nitrocellulose membrane by semidry blotting. On the membrane, the band corresponding to asFP595 appears red under daylight conditions and exhibits orange fluorescence with an emission maximum at 595 nm under UV light (Fig. 4 A and C). The fluorescence is stable and can even be detected after passage of the renatured protein through the membrane because of extended blotting. Renaturation with full restoration of fluorescence in the presence of β-mercaptoethanol has been described for heat-denatured GFP (31). Surprisingly, asCP562, which is nonfluorescent in vivo, behaves in the same manner and shows identical fluorescence as renatured asFP595 (Fig. 4 A and C).


31. Surpin, M. A. & Ward, W. W. (1989) Photochem. Photobiol. 49, 65.

Anmerkungen

The source is mentioned three times, but it is not clear to the reader that the entire page is taken from it.

Sichter
(SleepyHollow02), Hindemith


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