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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).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.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.


Related in: MedlinePlus

Two-input transcriptional regulation offragmented IFP. The near-infraredfluorescence of E. coli (XL1-Blue)transformed with vectors that use IPTG- and arabinose-inducible promotersto express the N- and C-terminal fragments of IFP, respectively. Theindividual and combined effects of 5 mM arabinose (ARA) and 5 mM IPTGon whole cell fluorescence (λex = 684 nm; λem = 710 nm) was measured at 37 °C, normalized to celldensity, and reported relative to the fragmented IFP with the largestsignal. Error bars represent ±1σ.
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fig3: Two-input transcriptional regulation offragmented IFP. The near-infraredfluorescence of E. coli (XL1-Blue)transformed with vectors that use IPTG- and arabinose-inducible promotersto express the N- and C-terminal fragments of IFP, respectively. Theindividual and combined effects of 5 mM arabinose (ARA) and 5 mM IPTGon whole cell fluorescence (λex = 684 nm; λem = 710 nm) was measured at 37 °C, normalized to celldensity, and reported relative to the fragmented IFP with the largestsignal. Error bars represent ±1σ.

Mentions: The IFP BV chromophore makesa large number of noncovalent interactionswith residues within the C-terminal GAF domain that reside withinthe C-terminal fragments. This clustering of BV-interacting residuessuggested that fragmented IFP may require BV association with onlyone fragment to fluoresce. To test this idea, we cloned each pairof IFP gene fragments into vectors that use different inducible promoters(PT5 and PBAD) to control expression (Figure 3) and examined whether the maximum signal requiredexpression of both the N- and C-terminal fragments. Whole cell fluorescencemeasurements revealed that each of the fragmented IFP displayed thehighest fluorescence when E. coli harboringthese vectors was grown in the presence of both arabinose and IPTG.140-EK displayed the highest fluorescence, similar to that observedwith the constructs discovered in the EK library, and similar wholecell fluorescence as cells expressing full-length IFP from eitherinducible promoter (data not shown). In all cases, we detected a lowlevel of near-infrared fluorescence with cells grown only in the presenceof arabinose. This trend was interpreted as arising from basal expressionof the fragments under PT5 promoter regulation, which hasbeen observed previously with the vectors used for the measurements.32 These results show that all seven of the fragmentedIFP exhibit AND gate genetic logic.


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)

Two-input transcriptional regulation offragmented IFP. The near-infraredfluorescence of E. coli (XL1-Blue)transformed with vectors that use IPTG- and arabinose-inducible promotersto express the N- and C-terminal fragments of IFP, respectively. Theindividual and combined effects of 5 mM arabinose (ARA) and 5 mM IPTGon whole cell fluorescence (λex = 684 nm; λem = 710 nm) was measured at 37 °C, normalized to celldensity, and reported relative to the fragmented IFP with the largestsignal. Error bars represent ±1σ.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Two-input transcriptional regulation offragmented IFP. The near-infraredfluorescence of E. coli (XL1-Blue)transformed with vectors that use IPTG- and arabinose-inducible promotersto express the N- and C-terminal fragments of IFP, respectively. Theindividual and combined effects of 5 mM arabinose (ARA) and 5 mM IPTGon whole cell fluorescence (λex = 684 nm; λem = 710 nm) was measured at 37 °C, normalized to celldensity, and reported relative to the fragmented IFP with the largestsignal. Error bars represent ±1σ.
Mentions: The IFP BV chromophore makesa large number of noncovalent interactionswith residues within the C-terminal GAF domain that reside withinthe C-terminal fragments. This clustering of BV-interacting residuessuggested that fragmented IFP may require BV association with onlyone fragment to fluoresce. To test this idea, we cloned each pairof IFP gene fragments into vectors that use different inducible promoters(PT5 and PBAD) to control expression (Figure 3) and examined whether the maximum signal requiredexpression of both the N- and C-terminal fragments. Whole cell fluorescencemeasurements revealed that each of the fragmented IFP displayed thehighest fluorescence when E. coli harboringthese vectors was grown in the presence of both arabinose and IPTG.140-EK displayed the highest fluorescence, similar to that observedwith the constructs discovered in the EK library, and similar wholecell fluorescence as cells expressing full-length IFP from eitherinducible promoter (data not shown). In all cases, we detected a lowlevel of near-infrared fluorescence with cells grown only in the presenceof arabinose. This trend was interpreted as arising from basal expressionof the fragments under PT5 promoter regulation, which hasbeen observed previously with the vectors used for the measurements.32 These results show that all seven of the fragmentedIFP exhibit AND gate genetic logic.

Bottom Line: 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.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.


Related in: MedlinePlus