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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.


Removalof IAAL-E3 decreases IFP fragment complementation. (A)Fluorescence of E. coli (λex = 684 nm; λem = 710 nm) expressing fragmentedIFP with their C-terminal fragments fused to IAAL-K3 and their N-terminalfragments lacking a peptide. Fluorescence is reported as the fractionof the signal observed with full-length IFP. (B) Ratio of fluorescencemeasured for fragmented IFP having IAAL-K3 fused at the terminus ofone fragment (+K) to fragmented IFP homologues lacking peptide fusions(−K). The fluorescence intensity of split variants containingone K coil was not significantly different from that of variants lackingboth coils, with the exception of variants 142 and 144 (two tailed t-test; p < 0.05). (C) Western blotdetection of N-terminal IFP fragments (IFP-F1), which have a GST tag,and the C-terminal fragments fused to IAAL-K3 (IFP-F2 + K3), whichhave an HA tag. Error bars represent ±1σ.
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fig5: Removalof IAAL-E3 decreases IFP fragment complementation. (A)Fluorescence of E. coli (λex = 684 nm; λem = 710 nm) expressing fragmentedIFP with their C-terminal fragments fused to IAAL-K3 and their N-terminalfragments lacking a peptide. Fluorescence is reported as the fractionof the signal observed with full-length IFP. (B) Ratio of fluorescencemeasured for fragmented IFP having IAAL-K3 fused at the terminus ofone fragment (+K) to fragmented IFP homologues lacking peptide fusions(−K). The fluorescence intensity of split variants containingone K coil was not significantly different from that of variants lackingboth coils, with the exception of variants 142 and 144 (two tailed t-test; p < 0.05). (C) Western blotdetection of N-terminal IFP fragments (IFP-F1), which have a GST tag,and the C-terminal fragments fused to IAAL-K3 (IFP-F2 + K3), whichhave an HA tag. Error bars represent ±1σ.

Mentions: We posited that the decreasedexpression of C-terminal fragmentsupon IAAL-K3 removal arose in part because translation initiationof this fragment was decreased by changing the context of the RBSdriving expression. This idea was tested by using a thermodynamicmodel to calculate the relative translation initiation rates fromthe RBS controlling IFP fragment expression.33 All seven of the C-terminal fragments had lower calculated translationinitiation rates upon removal of the IAAL-K3 peptide (Figure S5). To test this prediction, we createdvectors that expressed all of the IFP variants with only one fragment(C-terminal) fused to a peptide (IAAL-K3). Thermodynamic calculationspredicted that all of these vectors would initiate translation ofthe IFP fragments to the same extent as that of fragmented IFP fusedto both IAAL-E3 and IAAL-K3. We found that fragmented IFP fused toonly one peptide displayed 1–7.5% of the whole cell fluorescenceobserved with full-length IFP (Figure 5A).This fluorescence emission was similar to or lower than that observedwith homologous IFP lacking both peptides (Figure 5B). We also found that the excitation and emission spectrumof each variant was comparable to that of IFP fragments fused to IAAL-E3and IAAL-K3 (Table S1). Western blot analysisdid not reveal a strong correlation between near-infrared emissionand the relative expression of the different N- and C-terminal fragments(Figure 5C). However, we found that the C-terminalfragments fused to the IAAL-K3 peptide accumulated to greater levelsthan the same IFP fragments lacking the peptide (Figure S4), as predicted from our RBS strength analysis.


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)

Removalof IAAL-E3 decreases IFP fragment complementation. (A)Fluorescence of E. coli (λex = 684 nm; λem = 710 nm) expressing fragmentedIFP with their C-terminal fragments fused to IAAL-K3 and their N-terminalfragments lacking a peptide. Fluorescence is reported as the fractionof the signal observed with full-length IFP. (B) Ratio of fluorescencemeasured for fragmented IFP having IAAL-K3 fused at the terminus ofone fragment (+K) to fragmented IFP homologues lacking peptide fusions(−K). The fluorescence intensity of split variants containingone K coil was not significantly different from that of variants lackingboth coils, with the exception of variants 142 and 144 (two tailed t-test; p < 0.05). (C) Western blotdetection of N-terminal IFP fragments (IFP-F1), which have a GST tag,and the C-terminal fragments fused to IAAL-K3 (IFP-F2 + K3), whichhave an HA tag. Error bars represent ±1σ.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4487222&req=5

fig5: Removalof IAAL-E3 decreases IFP fragment complementation. (A)Fluorescence of E. coli (λex = 684 nm; λem = 710 nm) expressing fragmentedIFP with their C-terminal fragments fused to IAAL-K3 and their N-terminalfragments lacking a peptide. Fluorescence is reported as the fractionof the signal observed with full-length IFP. (B) Ratio of fluorescencemeasured for fragmented IFP having IAAL-K3 fused at the terminus ofone fragment (+K) to fragmented IFP homologues lacking peptide fusions(−K). The fluorescence intensity of split variants containingone K coil was not significantly different from that of variants lackingboth coils, with the exception of variants 142 and 144 (two tailed t-test; p < 0.05). (C) Western blotdetection of N-terminal IFP fragments (IFP-F1), which have a GST tag,and the C-terminal fragments fused to IAAL-K3 (IFP-F2 + K3), whichhave an HA tag. Error bars represent ±1σ.
Mentions: We posited that the decreasedexpression of C-terminal fragmentsupon IAAL-K3 removal arose in part because translation initiationof this fragment was decreased by changing the context of the RBSdriving expression. This idea was tested by using a thermodynamicmodel to calculate the relative translation initiation rates fromthe RBS controlling IFP fragment expression.33 All seven of the C-terminal fragments had lower calculated translationinitiation rates upon removal of the IAAL-K3 peptide (Figure S5). To test this prediction, we createdvectors that expressed all of the IFP variants with only one fragment(C-terminal) fused to a peptide (IAAL-K3). Thermodynamic calculationspredicted that all of these vectors would initiate translation ofthe IFP fragments to the same extent as that of fragmented IFP fusedto both IAAL-E3 and IAAL-K3. We found that fragmented IFP fused toonly one peptide displayed 1–7.5% of the whole cell fluorescenceobserved with full-length IFP (Figure 5A).This fluorescence emission was similar to or lower than that observedwith homologous IFP lacking both peptides (Figure 5B). We also found that the excitation and emission spectrumof each variant was comparable to that of IFP fragments fused to IAAL-E3and IAAL-K3 (Table S1). Western blot analysisdid not reveal a strong correlation between near-infrared emissionand the relative expression of the different N- and C-terminal fragments(Figure 5C). However, we found that the C-terminalfragments fused to the IAAL-K3 peptide accumulated to greater levelsthan the same IFP fragments lacking the peptide (Figure S4), as predicted from our RBS strength analysis.

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.