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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

Fluorescentfragmented IFP from the EK library. (A) For those variantsthat displayed near-infrared fluorescence, the IFP backbone fissionsites (spheres) were mapped onto the PAS (blue) and GAF (red) domainsof BphP, PDB = 1ZTU,25 using PyMol. Each site representsthe last IFP residue in the N-terminal fragment. The domain linkerthat is disordered in the BphP structure is shown as a dashed line.(B) Fluorescence of each fragmented IFP upon expression in E. coli is shown relative to that of cells expressingfull-length IFP and lacking an IFP (−ctrl). Whole cell fluorescence(λex = 684 nm; λem = 710 nm) wasmeasured at 37 °C and normalized to cell density. The fluorescenceintensity obtained with cells expressing each split variant was significantlydifferent from cells lacking an IFP (two tailed t-test; p < 0.01). (C) Western blot detectionof N-terminal IFP fragments fused to IAAL-E3 (IFP-F1 + E3), whichhave a GST tag, and the C-terminal IFP fragments fused to IAAL-K3(IFP-F2 + K3), which have an HA tag. Error bars represent ±1σcalculated using three or more replicates.
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fig2: Fluorescentfragmented IFP from the EK library. (A) For those variantsthat displayed near-infrared fluorescence, the IFP backbone fissionsites (spheres) were mapped onto the PAS (blue) and GAF (red) domainsof BphP, PDB = 1ZTU,25 using PyMol. Each site representsthe last IFP residue in the N-terminal fragment. The domain linkerthat is disordered in the BphP structure is shown as a dashed line.(B) Fluorescence of each fragmented IFP upon expression in E. coli is shown relative to that of cells expressingfull-length IFP and lacking an IFP (−ctrl). Whole cell fluorescence(λex = 684 nm; λem = 710 nm) wasmeasured at 37 °C and normalized to cell density. The fluorescenceintensity obtained with cells expressing each split variant was significantlydifferent from cells lacking an IFP (two tailed t-test; p < 0.01). (C) Western blot detectionof N-terminal IFP fragments fused to IAAL-E3 (IFP-F1 + E3), whichhave a GST tag, and the C-terminal IFP fragments fused to IAAL-K3(IFP-F2 + K3), which have an HA tag. Error bars represent ±1σcalculated using three or more replicates.

Mentions: Screening1760 clones from the EK library identified multiple colonies withλem > 700 nm, and sequencing vectors from thesecoloniesidentified seven unique fragmented IFP having their peptide backbonecleaved distal (>25 residues) from the termini. Mapping the locationsof these backbone cleavage sites onto the D. radiodurans BphP chromophore-binding domain,25 theprotein that was mutated to create IFP,24 revealed that all of the fission sites are proximal to the linkerthat connects the PAS (Per/ARNT/Sim) and GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA) domains within IFP (Figure 2A). These fission sites occur at the end of the second β-sheetwithin the PAS domain (117-EK and 118-EK), the end of the third β-sheetwithin the PAS domain (129-EK and 131-EK), and the beginning of firsthelix within the GAF domain (140-EK, 142-EK, and 144-EK).


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)

Fluorescentfragmented IFP from the EK library. (A) For those variantsthat displayed near-infrared fluorescence, the IFP backbone fissionsites (spheres) were mapped onto the PAS (blue) and GAF (red) domainsof BphP, PDB = 1ZTU,25 using PyMol. Each site representsthe last IFP residue in the N-terminal fragment. The domain linkerthat is disordered in the BphP structure is shown as a dashed line.(B) Fluorescence of each fragmented IFP upon expression in E. coli is shown relative to that of cells expressingfull-length IFP and lacking an IFP (−ctrl). Whole cell fluorescence(λex = 684 nm; λem = 710 nm) wasmeasured at 37 °C and normalized to cell density. The fluorescenceintensity obtained with cells expressing each split variant was significantlydifferent from cells lacking an IFP (two tailed t-test; p < 0.01). (C) Western blot detectionof N-terminal IFP fragments fused to IAAL-E3 (IFP-F1 + E3), whichhave a GST tag, and the C-terminal IFP fragments fused to IAAL-K3(IFP-F2 + K3), which have an HA tag. Error bars represent ±1σcalculated using three or more replicates.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Fluorescentfragmented IFP from the EK library. (A) For those variantsthat displayed near-infrared fluorescence, the IFP backbone fissionsites (spheres) were mapped onto the PAS (blue) and GAF (red) domainsof BphP, PDB = 1ZTU,25 using PyMol. Each site representsthe last IFP residue in the N-terminal fragment. The domain linkerthat is disordered in the BphP structure is shown as a dashed line.(B) Fluorescence of each fragmented IFP upon expression in E. coli is shown relative to that of cells expressingfull-length IFP and lacking an IFP (−ctrl). Whole cell fluorescence(λex = 684 nm; λem = 710 nm) wasmeasured at 37 °C and normalized to cell density. The fluorescenceintensity obtained with cells expressing each split variant was significantlydifferent from cells lacking an IFP (two tailed t-test; p < 0.01). (C) Western blot detectionof N-terminal IFP fragments fused to IAAL-E3 (IFP-F1 + E3), whichhave a GST tag, and the C-terminal IFP fragments fused to IAAL-K3(IFP-F2 + K3), which have an HA tag. Error bars represent ±1σcalculated using three or more replicates.
Mentions: Screening1760 clones from the EK library identified multiple colonies withλem > 700 nm, and sequencing vectors from thesecoloniesidentified seven unique fragmented IFP having their peptide backbonecleaved distal (>25 residues) from the termini. Mapping the locationsof these backbone cleavage sites onto the D. radiodurans BphP chromophore-binding domain,25 theprotein that was mutated to create IFP,24 revealed that all of the fission sites are proximal to the linkerthat connects the PAS (Per/ARNT/Sim) and GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA) domains within IFP (Figure 2A). These fission sites occur at the end of the second β-sheetwithin the PAS domain (117-EK and 118-EK), the end of the third β-sheetwithin the PAS domain (129-EK and 131-EK), and the beginning of firsthelix within the GAF domain (140-EK, 142-EK, and 144-EK).

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