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Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

Pandey N, Nobles CL, Zechiedrich L, Maresso AW, Silberg JJ - ACS Synth Biol (2014)

Bottom Line: However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP).Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker.Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence.

View Article: PubMed Central - PubMed

Affiliation: †Department of Biosciences, Rice University, Houston, Texas 77005, United States.

ABSTRACT
Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals.

No MeSH data available.


CheAand CheY rescue IFP fragment complementation. (A) Whole cellfluorescence of E. coli expressingeach pair of IFP fragments as fusions to CheA and CheY. Fluorescenceemission (λex = 684 nm; λem = 710nm) measured at 37 °C was normalized to cell density and is shownrelative to the signal from cells expressing full-length IFP. (B)Ratio of fluorescence measured for fragmented IFP having CheA andCheY fused at their termini (+AY) to that of homologous IFP fragmentslacking CheA and CheY (−AY). The fluorescence intensity obtainedwith each split variant +AY was significantly different from thatfor homologous variants −AY (two tailed t-test; p < 0.005). (C) Ratio of fluorescence for fragmentedIFP having IAAL-E3 and IAAL-K3 fused at their termini (+EK) to thatfor homologous IFP fragments having CheA and CheY fused at their termini(+AY). Error bars represent ±1σ. Four variants (118, 140,142, and 144) displayed significantly higher fluorescence +EK comparedwith that +AY (two tailed t-test; p < 0.05).
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fig6: CheAand CheY rescue IFP fragment complementation. (A) Whole cellfluorescence of E. coli expressingeach pair of IFP fragments as fusions to CheA and CheY. Fluorescenceemission (λex = 684 nm; λem = 710nm) measured at 37 °C was normalized to cell density and is shownrelative to the signal from cells expressing full-length IFP. (B)Ratio of fluorescence measured for fragmented IFP having CheA andCheY fused at their termini (+AY) to that of homologous IFP fragmentslacking CheA and CheY (−AY). The fluorescence intensity obtainedwith each split variant +AY was significantly different from thatfor homologous variants −AY (two tailed t-test; p < 0.005). (C) Ratio of fluorescence for fragmentedIFP having IAAL-E3 and IAAL-K3 fused at their termini (+EK) to thatfor homologous IFP fragments having CheA and CheY fused at their termini(+AY). Error bars represent ±1σ. Four variants (118, 140,142, and 144) displayed significantly higher fluorescence +EK comparedwith that +AY (two tailed t-test; p < 0.05).

Mentions: We found that IFP fragmentsfused to CheA and CheY displayed between15 and 71% of the whole cell fluorescence observed with full-lengthIFP (Figure 6A). CheA and CheY enhanced IFPfragment complementation to varying extents over IFP fragments lackingfusions to interacting proteins, ranging from 4- to 22-fold (Figure 6B). The relative effects of CheA and CheY on fragmentedIFP emission were also similar to the effects of IAAL-E3 and IAAL-K3.IFP fragmented after residues 117 and 144 displayed the largest fluorescenceenhancement upon fusion to the associating proteins, whereas IFP fragmentedafter residues 129 and 142 consistently exhibited the smallest enhancement.On average, however, fragmented IFP fused to CheA and CheY displayedlower whole cell fluorescence than that of the same IFP fragmentsfused to IAAL-E3 and IAAL-K3 (Figure 6C). Thesedifferences were not interpreted as arising from changes in spectralproperties or fragment expression. Fragmented IFP fused to CheA andCheY displayed spectra with similar excitation and emission maximaas that of IFP (Table S1), and IFP fragmentsaccumulated to a similar level when fused to CheA/CheY and IAAL-E3/IAAL-K3(Figure S4). Instead, these differencesare thought to arise from the varying strength of the protein–proteininteractions used to assist with IFP fragment complementation. Thefragment complementation correlated with the relative affinities ofthe CheA/CheY (KD = 200 nM) and IAAL-E3/IAAL-K3(KD = 70 nM) complexes.30,34


Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

Pandey N, Nobles CL, Zechiedrich L, Maresso AW, Silberg JJ - ACS Synth Biol (2014)

CheAand CheY rescue IFP fragment complementation. (A) Whole cellfluorescence of E. coli expressingeach pair of IFP fragments as fusions to CheA and CheY. Fluorescenceemission (λex = 684 nm; λem = 710nm) measured at 37 °C was normalized to cell density and is shownrelative to the signal from cells expressing full-length IFP. (B)Ratio of fluorescence measured for fragmented IFP having CheA andCheY fused at their termini (+AY) to that of homologous IFP fragmentslacking CheA and CheY (−AY). The fluorescence intensity obtainedwith each split variant +AY was significantly different from thatfor homologous variants −AY (two tailed t-test; p < 0.005). (C) Ratio of fluorescence for fragmentedIFP having IAAL-E3 and IAAL-K3 fused at their termini (+EK) to thatfor homologous IFP fragments having CheA and CheY fused at their termini(+AY). Error bars represent ±1σ. Four variants (118, 140,142, and 144) displayed significantly higher fluorescence +EK comparedwith that +AY (two tailed t-test; p < 0.05).
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fig6: CheAand CheY rescue IFP fragment complementation. (A) Whole cellfluorescence of E. coli expressingeach pair of IFP fragments as fusions to CheA and CheY. Fluorescenceemission (λex = 684 nm; λem = 710nm) measured at 37 °C was normalized to cell density and is shownrelative to the signal from cells expressing full-length IFP. (B)Ratio of fluorescence measured for fragmented IFP having CheA andCheY fused at their termini (+AY) to that of homologous IFP fragmentslacking CheA and CheY (−AY). The fluorescence intensity obtainedwith each split variant +AY was significantly different from thatfor homologous variants −AY (two tailed t-test; p < 0.005). (C) Ratio of fluorescence for fragmentedIFP having IAAL-E3 and IAAL-K3 fused at their termini (+EK) to thatfor homologous IFP fragments having CheA and CheY fused at their termini(+AY). Error bars represent ±1σ. Four variants (118, 140,142, and 144) displayed significantly higher fluorescence +EK comparedwith that +AY (two tailed t-test; p < 0.05).
Mentions: We found that IFP fragmentsfused to CheA and CheY displayed between15 and 71% of the whole cell fluorescence observed with full-lengthIFP (Figure 6A). CheA and CheY enhanced IFPfragment complementation to varying extents over IFP fragments lackingfusions to interacting proteins, ranging from 4- to 22-fold (Figure 6B). The relative effects of CheA and CheY on fragmentedIFP emission were also similar to the effects of IAAL-E3 and IAAL-K3.IFP fragmented after residues 117 and 144 displayed the largest fluorescenceenhancement upon fusion to the associating proteins, whereas IFP fragmentedafter residues 129 and 142 consistently exhibited the smallest enhancement.On average, however, fragmented IFP fused to CheA and CheY displayedlower whole cell fluorescence than that of the same IFP fragmentsfused to IAAL-E3 and IAAL-K3 (Figure 6C). Thesedifferences were not interpreted as arising from changes in spectralproperties or fragment expression. Fragmented IFP fused to CheA andCheY displayed spectra with similar excitation and emission maximaas that of IFP (Table S1), and IFP fragmentsaccumulated to a similar level when fused to CheA/CheY and IAAL-E3/IAAL-K3(Figure S4). Instead, these differencesare thought to arise from the varying strength of the protein–proteininteractions used to assist with IFP fragment complementation. Thefragment complementation correlated with the relative affinities ofthe CheA/CheY (KD = 200 nM) and IAAL-E3/IAAL-K3(KD = 70 nM) complexes.30,34

Bottom Line: However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP).Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker.Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence.

View Article: PubMed Central - PubMed

Affiliation: †Department of Biosciences, Rice University, Houston, Texas 77005, United States.

ABSTRACT
Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals.

No MeSH data available.