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Laboratory evolution of fast-folding green fluorescent protein using secretory pathway quality control.

Fisher AC, DeLisa MP - PLoS ONE (2008)

Bottom Line: Recently, a superfolder GFP was engineered with increased resistance to denaturation and improved folding kinetics.Each of these GFP variants exhibited much faster folding kinetics than the parental GFPmut2 protein and one of these, designated superfast GFP, folded at a rate that even exceeded superfolder GFP.Remarkably, these GFP variants exhibited little to no loss in specific fluorescence activity relative to GFPmut2, suggesting that the process of superfolding can be accomplished without altering the proteins' normal function.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Green fluorescent protein (GFP) has undergone a long history of optimization to become one of the most popular proteins in all of cell biology. It is thermally and chemically robust and produces a pronounced fluorescent phenotype when expressed in cells of all types. Recently, a superfolder GFP was engineered with increased resistance to denaturation and improved folding kinetics. Here we report that unlike other well-folded variants of GFP (e.g., GFPmut2), superfolder GFP was spared from elimination when targeted for secretion via the SecYEG translocase. This prompted us to hypothesize that the folding quality control inherent to this secretory pathway could be used as a platform for engineering similar 'superfolded' proteins. To test this, we targeted a combinatorial library of GFPmut2 variants to the SecYEG translocase and isolated several superfolded variants that accumulated in the cytoplasm due to their enhanced folding properties. Each of these GFP variants exhibited much faster folding kinetics than the parental GFPmut2 protein and one of these, designated superfast GFP, folded at a rate that even exceeded superfolder GFP. Remarkably, these GFP variants exhibited little to no loss in specific fluorescence activity relative to GFPmut2, suggesting that the process of superfolding can be accomplished without altering the proteins' normal function. Overall, we demonstrate that laboratory evolution combined with secretory pathway quality control enables sampling of largely unexplored amino-acid sequences for the discovery of artificial, high-performance proteins with properties that are unparalleled in their naturally occurring analogues.

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In vitro characterization of GFP folding.(a) Kinetic refolding measured as fraction folded (Ff) over time, (b) equilibrium unfolding measured as Ff versus GdnHCl molarity and (c) kinetic unfolding measured as Ff over time for the different GFP variants as indicated. Curve fits were added as a visual aid. All data is the average of three replicate experiments where the standard error for all data was <10%.
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pone-0002351-g003: In vitro characterization of GFP folding.(a) Kinetic refolding measured as fraction folded (Ff) over time, (b) equilibrium unfolding measured as Ff versus GdnHCl molarity and (c) kinetic unfolding measured as Ff over time for the different GFP variants as indicated. Curve fits were added as a visual aid. All data is the average of three replicate experiments where the standard error for all data was <10%.

Mentions: We next chose to characterize clones P4, P5 and P7 in greater detail because cells expressing ssDsbA fused to each yielded the three highest whole-cell fluorescence values and collectively contained six of the seven recurring substitutions mentioned above. Following purification, we observed that P4, P5, and P7 had a slightly lower soluble yield and slightly lower total fluorescence relative to GFPmut2 (Table 1), suggesting that these clones represent a novel solution to protein folding optimization as neither the function nor the soluble yield of the protein-of-interest was improved. Since it has been shown in the past that fast-folding proteins can be trapped in the cytoplasm during Sec transport [30] and since sfGFP folds faster than frGFP [7], we reasoned that a logical explanation for the cytoplasmic accumulation of P4, P5, and P7 was increased folding kinetics. Indeed, following complete unfolding, the refolding speed of variants P4 and P5 was comparable to, while P7 far eclipsed, that of sfGFP (Fig. 3a, Table 1). Since sfGFP was one of the fastest folding GFPs to date [7], we renamed P7 ‘superfast’ GFP. In addition, P4, P5, superfast GFP, and sfGFP all approached complete recovery during refolding while frGFP and GFPmut2 stalled around 60% and 75% recovery, respectively.


Laboratory evolution of fast-folding green fluorescent protein using secretory pathway quality control.

Fisher AC, DeLisa MP - PLoS ONE (2008)

In vitro characterization of GFP folding.(a) Kinetic refolding measured as fraction folded (Ff) over time, (b) equilibrium unfolding measured as Ff versus GdnHCl molarity and (c) kinetic unfolding measured as Ff over time for the different GFP variants as indicated. Curve fits were added as a visual aid. All data is the average of three replicate experiments where the standard error for all data was <10%.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2396501&req=5

pone-0002351-g003: In vitro characterization of GFP folding.(a) Kinetic refolding measured as fraction folded (Ff) over time, (b) equilibrium unfolding measured as Ff versus GdnHCl molarity and (c) kinetic unfolding measured as Ff over time for the different GFP variants as indicated. Curve fits were added as a visual aid. All data is the average of three replicate experiments where the standard error for all data was <10%.
Mentions: We next chose to characterize clones P4, P5 and P7 in greater detail because cells expressing ssDsbA fused to each yielded the three highest whole-cell fluorescence values and collectively contained six of the seven recurring substitutions mentioned above. Following purification, we observed that P4, P5, and P7 had a slightly lower soluble yield and slightly lower total fluorescence relative to GFPmut2 (Table 1), suggesting that these clones represent a novel solution to protein folding optimization as neither the function nor the soluble yield of the protein-of-interest was improved. Since it has been shown in the past that fast-folding proteins can be trapped in the cytoplasm during Sec transport [30] and since sfGFP folds faster than frGFP [7], we reasoned that a logical explanation for the cytoplasmic accumulation of P4, P5, and P7 was increased folding kinetics. Indeed, following complete unfolding, the refolding speed of variants P4 and P5 was comparable to, while P7 far eclipsed, that of sfGFP (Fig. 3a, Table 1). Since sfGFP was one of the fastest folding GFPs to date [7], we renamed P7 ‘superfast’ GFP. In addition, P4, P5, superfast GFP, and sfGFP all approached complete recovery during refolding while frGFP and GFPmut2 stalled around 60% and 75% recovery, respectively.

Bottom Line: Recently, a superfolder GFP was engineered with increased resistance to denaturation and improved folding kinetics.Each of these GFP variants exhibited much faster folding kinetics than the parental GFPmut2 protein and one of these, designated superfast GFP, folded at a rate that even exceeded superfolder GFP.Remarkably, these GFP variants exhibited little to no loss in specific fluorescence activity relative to GFPmut2, suggesting that the process of superfolding can be accomplished without altering the proteins' normal function.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Green fluorescent protein (GFP) has undergone a long history of optimization to become one of the most popular proteins in all of cell biology. It is thermally and chemically robust and produces a pronounced fluorescent phenotype when expressed in cells of all types. Recently, a superfolder GFP was engineered with increased resistance to denaturation and improved folding kinetics. Here we report that unlike other well-folded variants of GFP (e.g., GFPmut2), superfolder GFP was spared from elimination when targeted for secretion via the SecYEG translocase. This prompted us to hypothesize that the folding quality control inherent to this secretory pathway could be used as a platform for engineering similar 'superfolded' proteins. To test this, we targeted a combinatorial library of GFPmut2 variants to the SecYEG translocase and isolated several superfolded variants that accumulated in the cytoplasm due to their enhanced folding properties. Each of these GFP variants exhibited much faster folding kinetics than the parental GFPmut2 protein and one of these, designated superfast GFP, folded at a rate that even exceeded superfolder GFP. Remarkably, these GFP variants exhibited little to no loss in specific fluorescence activity relative to GFPmut2, suggesting that the process of superfolding can be accomplished without altering the proteins' normal function. Overall, we demonstrate that laboratory evolution combined with secretory pathway quality control enables sampling of largely unexplored amino-acid sequences for the discovery of artificial, high-performance proteins with properties that are unparalleled in their naturally occurring analogues.

Show MeSH