The DOI matches its Crossref record; 25 of the 25 references it cites were found in a registry or an index.
Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein
Abstract
The green-fluorescent protein (GFP) that functions as a bioluminescence energy transfer acceptor in the jellyfish Aequorea has been renatured with up to 90% yield following acid, base, or guanidine denaturation. Renaturation, following pH neutralization or simple dilution of guanidine, proceeds with a half-recovery time of less than 5 min as measured by the return of visible fluorescence. Residual unrenatured protein has been quantitatively removed by chromatography on Sephadex G-75. The chromatographed, renatured GFP has corrected fluorescence excitation and emission spectra identical with those of the native protein at pH 7.0 (excitation lambda max = 398 nm; emission lambda max = 508 nm) and also at pH 12.2 (excitation lambda max = 476 nm; emission lambda max = 505 nm). With its peak position red-shifted 78 nm at pH 12.2, the Aequorea GFP excitation spectrum more closely resembles the excitation spectra of Renilla (sea pansy) and Phialidium (hydromedusan) GFPs at neutral pH. Visible absorption spectra of the native and renatured Aequorea green-fluorescent proteins at pH 7.0 are also identical, suggesting that the chromophore binding site has returned to its native state. Small differences in far-UV absorption and circular dichroism spectra, however, indicate that the renatured protein has not fully regained its native secondary structure.
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Cites
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Renaturation of Aequorea green-fluorescent protein
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Self-citationThe spectral clustering of visual pigments
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Cited by
Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues
Chudakov, D. M., Matz, M. V., Lukyanov, S., & Lukyanov, K. A. (2010) · Physiological Reviews · doi:10.1152/physrev.00038.2009 · Cited by 1,375
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Haupts, U., Maiti, S., Schwille, P., & Webb, W. W. (1998) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.95.23.13573 · Cited by 554
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Metadata
- Authors
- Ward, W. W., & Bokman, S. H.
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 1982-09-14
- Volume
- 21
- Issue
- 19
- Pages
- 4535-4540
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W2053921060
- Cited by
- 342
- References
- 25
- FWCI
- 0.39
- Type
- journal article
- Language
- en
- Open access
- closed
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