Limits...
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.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.

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
Flow cytometric analysis of GFP variants.Fluorescence histograms for cells expressing: superfolder GFP (sf, gray fill), folding reporter GFP (fr, gray line), and GFPmut2 (mut2, black line) (a) without a signal peptide and a C-terminal 6xhis tag or (b) as an N-terminal fusion to the ssDsbA signal peptide; (c) an N-terminal ssDsbA signal peptide fused to GFPmut2 (black line) and TrxA-GFPmut2 (gray fill). The geometric mean is listed next to each histogram. There was no significant difference in growth rate between any of the cultures (data not shown). (d) Fluorescence (arbitrary units) and subcellular localization of GFP as measured for cytoplasmic (cyt, grey bars) and periplasmic (per, white bars) fractions generated from cells expressing the various ssDsbA-GFP fusions including sf, fr, mut2 and clones P1–P9. (e) Western blot analysis of the per and cyt fractions of cells expressing the same fusions probed with GFP antiserum. Blots were probed with anti-GroEL serum as a fractionation marker.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2396501&req=5

pone-0002351-g001: Flow cytometric analysis of GFP variants.Fluorescence histograms for cells expressing: superfolder GFP (sf, gray fill), folding reporter GFP (fr, gray line), and GFPmut2 (mut2, black line) (a) without a signal peptide and a C-terminal 6xhis tag or (b) as an N-terminal fusion to the ssDsbA signal peptide; (c) an N-terminal ssDsbA signal peptide fused to GFPmut2 (black line) and TrxA-GFPmut2 (gray fill). The geometric mean is listed next to each histogram. There was no significant difference in growth rate between any of the cultures (data not shown). (d) Fluorescence (arbitrary units) and subcellular localization of GFP as measured for cytoplasmic (cyt, grey bars) and periplasmic (per, white bars) fractions generated from cells expressing the various ssDsbA-GFP fusions including sf, fr, mut2 and clones P1–P9. (e) Western blot analysis of the per and cyt fractions of cells expressing the same fusions probed with GFP antiserum. Blots were probed with anti-GroEL serum as a fractionation marker.

Mentions: To facilitate directed evolution of superfolded proteins, our initial goal was to develop a cellular screen for easily differentiating between cells expressing frGFP and sfGFP. To begin, we examined cytoplasmic expression of frGFP and sfGFP. Despite the fact that the folding properties of sfGFP are far superior to those of frGFP, there was only a small increase (65%) in the geometric mean fluorescence of cells expressing sfGFP relative to frGFP (Fig. 1a). This increase was due mostly to an increase in the specific fluorescence of sfGFP rather than an increase in soluble expression for the better folding mutant (see Table 1). Since we desired a more pronounced difference in fluorescence emission between sfGFP and frGFP to facilitate directed evolution by fluorescence activated cell sorting (FACS), we explored whether protein quality control associated with either the Tat or SecYEG-mediated export pathways might be capable of yielding a more striking difference in fluorescence for sfGFP and frGFP. While the Tat pathway is known to modulate export efficiency of a substrate based on folding and solubility [18], [19], owing to the similar in vivo solubility profiles of sfGFP and frGFP we observed only a modest phenotypic difference between cells expressing sfGFP and frGFP when each was targeted to the Tat pathway (data not shown). Alternatively, we reasoned that proteins targeted for SecYEG export would experience one or more of the following fates: (a) accumulation in the periplasm if transport via SecYEG was successful; (b) degradation in the cytoplasm if transport failed and the protein was sensitive to proteolysis due to insufficient folding or stability; and (c) accumulation in the cytoplasm if transport failed but the protein was resistant to unfolding and proteolysis. Additionally, for GFP export, the latter scenario is the only one that would be expected to give rise to cellular fluorescence because GFP that is routed into the periplasm through SecYEG is non-fluorescent [31].


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

Fisher AC, DeLisa MP - PLoS ONE (2008)

Flow cytometric analysis of GFP variants.Fluorescence histograms for cells expressing: superfolder GFP (sf, gray fill), folding reporter GFP (fr, gray line), and GFPmut2 (mut2, black line) (a) without a signal peptide and a C-terminal 6xhis tag or (b) as an N-terminal fusion to the ssDsbA signal peptide; (c) an N-terminal ssDsbA signal peptide fused to GFPmut2 (black line) and TrxA-GFPmut2 (gray fill). The geometric mean is listed next to each histogram. There was no significant difference in growth rate between any of the cultures (data not shown). (d) Fluorescence (arbitrary units) and subcellular localization of GFP as measured for cytoplasmic (cyt, grey bars) and periplasmic (per, white bars) fractions generated from cells expressing the various ssDsbA-GFP fusions including sf, fr, mut2 and clones P1–P9. (e) Western blot analysis of the per and cyt fractions of cells expressing the same fusions probed with GFP antiserum. Blots were probed with anti-GroEL serum as a fractionation marker.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002351-g001: Flow cytometric analysis of GFP variants.Fluorescence histograms for cells expressing: superfolder GFP (sf, gray fill), folding reporter GFP (fr, gray line), and GFPmut2 (mut2, black line) (a) without a signal peptide and a C-terminal 6xhis tag or (b) as an N-terminal fusion to the ssDsbA signal peptide; (c) an N-terminal ssDsbA signal peptide fused to GFPmut2 (black line) and TrxA-GFPmut2 (gray fill). The geometric mean is listed next to each histogram. There was no significant difference in growth rate between any of the cultures (data not shown). (d) Fluorescence (arbitrary units) and subcellular localization of GFP as measured for cytoplasmic (cyt, grey bars) and periplasmic (per, white bars) fractions generated from cells expressing the various ssDsbA-GFP fusions including sf, fr, mut2 and clones P1–P9. (e) Western blot analysis of the per and cyt fractions of cells expressing the same fusions probed with GFP antiserum. Blots were probed with anti-GroEL serum as a fractionation marker.
Mentions: To facilitate directed evolution of superfolded proteins, our initial goal was to develop a cellular screen for easily differentiating between cells expressing frGFP and sfGFP. To begin, we examined cytoplasmic expression of frGFP and sfGFP. Despite the fact that the folding properties of sfGFP are far superior to those of frGFP, there was only a small increase (65%) in the geometric mean fluorescence of cells expressing sfGFP relative to frGFP (Fig. 1a). This increase was due mostly to an increase in the specific fluorescence of sfGFP rather than an increase in soluble expression for the better folding mutant (see Table 1). Since we desired a more pronounced difference in fluorescence emission between sfGFP and frGFP to facilitate directed evolution by fluorescence activated cell sorting (FACS), we explored whether protein quality control associated with either the Tat or SecYEG-mediated export pathways might be capable of yielding a more striking difference in fluorescence for sfGFP and frGFP. While the Tat pathway is known to modulate export efficiency of a substrate based on folding and solubility [18], [19], owing to the similar in vivo solubility profiles of sfGFP and frGFP we observed only a modest phenotypic difference between cells expressing sfGFP and frGFP when each was targeted to the Tat pathway (data not shown). Alternatively, we reasoned that proteins targeted for SecYEG export would experience one or more of the following fates: (a) accumulation in the periplasm if transport via SecYEG was successful; (b) degradation in the cytoplasm if transport failed and the protein was sensitive to proteolysis due to insufficient folding or stability; and (c) accumulation in the cytoplasm if transport failed but the protein was resistant to unfolding and proteolysis. Additionally, for GFP export, the latter scenario is the only one that would be expected to give rise to cellular fluorescence because GFP that is routed into the periplasm through SecYEG is non-fluorescent [31].

Bottom Line: Recently, a superfolder GFP was engineered with increased resistance to denaturation and improved folding kinetics.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.

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