Rapid Purification of Recombinant Green Fluorescent Protein Using the Hydrophobic Properties of an HPLC Size-Exclusion Column

Deschamps, Jeffrey R.; Miller, C. E.; Ward, Keith B.

doi:10.1006/prep.1995.1073

Cited by 50 publications

(32 citation statements)

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“…The bacteria cells were pelleted 6-12 h later and frozen in liquid nitrogen until purification. Proteins were purified aerobically by modifying a published protocol (26) to incorporate HQ (26 mm ϫ 30 cm) (PerSeptive Biosystems, Framingham, MA) and S-100 (26 mm ϫ 60 cm) (Pharmacia) columns (27). To prepare the Gly-Gly-Gly anaerobic sample, the protein was purified and crystallized in an anaerobic glove box (Vacuum Atmospheres, Hawthorne, CA).…”

Section: Methodsmentioning

confidence: 99%

Mechanism and energetics of green fluorescent protein chromophore synthesis revealed by trapped intermediate structures

Barondeau

Putnam

Kassmann

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

200

266

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Green fluorescent protein has revolutionized cell labeling and molecular tagging, yet the driving force and mechanism for its spontaneous fluorophore synthesis are not established. Here we discover mutations that substantially slow the rate but not the yield of this posttranslational modification, determine structures of the trapped precyclization intermediate and oxidized postcyclization states, and identify unanticipated features critical to chromophore maturation. The protein architecture contains a dramatic Ϸ80°bend in the central helix, which focuses distortions at G67 to promote ring formation from amino acids S65, Y66, and G67. Significantly, these distortions eliminate potential helical hydrogen bonds that would otherwise have to be broken at an energetic cost during peptide cyclization and force the G67 nitrogen and S65 carbonyl oxygen atoms within van der Waals contact in preparation for covalent bond formation. Further, we determine that under aerobic, but not anaerobic, conditions the Gly-Gly-Gly chromophore sequence cyclizes and incorporates an oxygen atom. These results lead directly to a conjugation-trapping mechanism, in which a thermodynamically unfavorable cyclization reaction is coupled to an electronic conjugation trapping step, to drive chromophore maturation. Moreover, we propose primarily electrostatic roles for the R96 and E222 side chains in chromophore formation and suggest that the T62 carbonyl oxygen is the base that initiates the dehydration reaction. Our molecular mechanism provides the basis for understanding and eventually controlling chromophore creation.T he Aequorea victoria green fluorescent protein (GFP) undergoes a remarkable posttranslational modification to create a chromophore out of its amino acids (S65, Y66, and G67) (1-3). GFP is small (238 aa), tolerates both N-and C-terminal fusions, and can be targeted to specific cellular locations (4). Synthesis of the GFP fluorophore occurs spontaneously after protein folding without cofactors or accessory proteins (5), making GFP-protein fusions tractable in a variety of organisms. GFP mutants and homologs exhibit fluorescent emission maxima ranging from blue to red (3, 6-8), which allow concurrent surveillance of multiple targets. Together, these properties have fundamentally altered in vivo molecular tagging and cell labeling. In addition, GFP-based indicators monitor cellular redox potential (9), pH (10, 11), metal ion concentrations (12, 13), and halide levels (14, 15). Because of these applications and the novel fluorophore, there have been extensive structural, spectroscopic, and biochemical characterizations of the protein and its mutants, all in the mature chromophore state (4, 16). The crystallographic structure of GFP reveals that the overall fold is an 11-stranded antiparallel ␤-barrel protein with the chromophore located near the geometric center of the barrel on a distorted ␣-helix (1, 17). Few molecular details are known about chromophore maturation.The proposed fluorophore formation mechanism entails three steps: ...

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Section: Methodsmentioning

confidence: 99%

Mechanism and energetics of green fluorescent protein chromophore synthesis revealed by trapped intermediate structures

Barondeau

Putnam

Kassmann

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

200

266

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“…Deschamps et al employed chromatographic techniques to purify recombinant GFP from an E. coli expression system (74). After creating extracts from E. coli, ammonium sulfate at 40% saturation was added, leaving GFP in the supernatant, and then increased to 70% saturation to precipitate GFP.…”

Section: Purificationmentioning

confidence: 99%

Seeing the Wood through the Trees: A Review of Techniques for Distinguishing Green Fluorescent Protein from Endogenous Autofluorescence

Billinton

Knight

2001

Analytical Biochemistry

462

382

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“…Protein concentrations were determined spectrophotometrically based on the procedure outlined by Gill and von Hippel (35), using the following extinction coefficients: ⑀ 276 ϭ 8700 M Ϫ1 cm Ϫ1 for GroEL; ⑀ 276 ϭ 1450 M Ϫ1 cm Ϫ1 for GroES. Recombinant green fluorescent protein GFP was purified as described (36).…”

Section: Methodsmentioning

confidence: 99%

High Salt-induced Conversion of Escherichia coliGroEL into a Fully Functional Thermophilic Chaperonin

Kusmierczyk

Martin

2000

Journal of Biological Chemistry

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The GroE chaperonin system can adapt to and function at various environmental folding conditions. To examine chaperonin-assisted protein folding at high salt concentrations, we characterized Escherichia coli GroE chaperonin activity in 1.2 M ammonium sulfate. Our data are consistent with GroEL undergoing a conformational change at this salt concentration, characterized by elevated ATPase activity and increased exposure of hydrophobic surface, as indicated by increased binding of the fluorophore bis-(5,5)-8-anilino-1-naphthalene sulfonic acid to the chaperonin. The presence of the salt results in increased substrate stringency and dependence on the full GroE system for release and productive folding of substrate proteins. Surprisingly, GroEL is fully functional as a thermophilic chaperonin in high concentrations of ammonium sulfate and is stable at temperatures up to 75°C. At these extreme conditions, GroEL can suppress aggregation and mediate refolding of non-native proteins.

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Rapid Purification of Recombinant Green Fluorescent Protein Using the Hydrophobic Properties of an HPLC Size-Exclusion Column

Cited by 50 publications

References 0 publications

Mechanism and energetics of green fluorescent protein chromophore synthesis revealed by trapped intermediate structures

Mechanism and energetics of green fluorescent protein chromophore synthesis revealed by trapped intermediate structures

Seeing the Wood through the Trees: A Review of Techniques for Distinguishing Green Fluorescent Protein from Endogenous Autofluorescence

High Salt-induced Conversion of Escherichia coliGroEL into a Fully Functional Thermophilic Chaperonin

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