Combining Random Gene Fission and Rational Gene Fusion To Discover Near-Infrared Fluorescent Protein Fragments That Report on Protein–Protein Interactions

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.

[1]  Chang‐Deng Hu,et al.  Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis , 2003, Nature Biotechnology.

[2]  Stephen W Michnick,et al.  Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions. , 2002, Nature biotechnology.

[3]  K. E. Lundin,et al.  Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression , 2014, Molecular therapy. Nucleic acids.

[4]  L. Zechiedrich,et al.  Supercoiled Minivector DNA resists shear forces associated with gene therapy delivery , 2011, Gene Therapy.

[5]  L. Serrano,et al.  Strategies for protein synthetic biology , 2010, Nucleic acids research.

[6]  Thomas J Magliery,et al.  Detecting protein-protein interactions with GFP-fragment reassembly , 2004, Nature Methods.

[7]  Melvin I Simon,et al.  In different organisms, the mode of interaction between two signaling proteins is not necessarily conserved. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[8]  R. Hodges,et al.  Designing heterodimeric two-stranded alpha-helical coiled-coils. Effects of hydrophobicity and alpha-helical propensity on protein folding, stability, and specificity. , 2002, The Journal of biological chemistry.

[9]  T. Kerppola.  Visualization of molecular interactions using bimolecular fluorescence complementation analysis: characteristics of protein fragment complementation. , 2009, Chemical Society reviews.

[10]  S. Fields,et al.  A novel genetic system to detect protein–protein interactions , 1989, Nature.

[11]  R. Hodges,et al.  NMR solution structure of a highly stable de novo heterodimeric coiled‐coil , 2004, Biopolymers.

[12]  Vladislav V Verkhusha,et al.  A near-infrared BiFC reporter for in vivo imaging of protein-protein interactions. , 2013, Chemistry & biology.

[13]  D. Ladant,et al.  A bacterial two-hybrid system based on a reconstituted signal transduction pathway. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[14]  S. Michnick,et al.  Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[15]  A. Varshavsky,et al.  Split ubiquitin as a sensor of protein interactions in vivo. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Chang‐Deng Hu,et al.  Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. , 2002, Molecular cell.

[17]  J. Silberg,et al.  Thermostability promotes the cooperative function of split adenylate kinases. , 2008, Protein engineering, design & selection : PEDS.

[18]  N. Zhao,et al.  Transfection of shRNA-encoding Minivector DNA of a few hundred base pairs to regulate gene expression in lymphoma cells , 2010, Gene Therapy.

[19]  R. Vierstra,et al.  A light-sensing knot revealed by the structure of the chromophore-binding domain of phytochrome , 2005, Nature.

[20]  S. Michnick,et al.  An infrared reporter to detect spatiotemporal dynamics of protein-protein interactions , 2014, Nature Methods.

[21]  Thomas H. Segall-Shapiro,et al.  Mesophilic and hyperthermophilic adenylate kinases differ in their tolerance to random fragmentation. , 2011, Journal of molecular biology.

[22]  Thomas H. Segall-Shapiro,et al.  A genetically encoded and gate for cell-targeted metabolic labeling of proteins. , 2013, Journal of the American Chemical Society.

[23]  David E. Paschon,et al.  Construction of protein fragment complementation libraries using incremental truncation. , 2004, Methods in enzymology.

[24]  David E. Paschon,et al.  Enhanced catalytic efficiency of aminoglycoside phosphotransferase (3')-IIa achieved through protein fragmentation and reassembly. , 2005, Journal of molecular biology.

[25]  Adam J. Meyer,et al.  A ‘resource allocator’ for transcription based on a highly fragmented T7 RNA polymerase , 2014, Molecular systems biology.

[26]  K. Matthews,et al.  Generation of an AraC-araBAD promoter-regulated T7 expression system. , 2000, Analytical biochemistry.

[27]  R. Vierstra,et al.  High Resolution Structure of Deinococcus Bacteriophytochrome Yields New Insights into Phytochrome Architecture and Evolution* , 2007, Journal of Biological Chemistry.

[28]  J. H. Shim,et al.  Combinatorial protein engineering by incremental truncation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Matthew R Bennett,et al.  Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants , 2013, Proceedings of the National Academy of Sciences.

[30]  Hau B. Nguyen,et al.  A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association , 2013, Scientific Reports.

[31]  Stephen W. Michnick,et al.  Universal strategies in research and drug discovery based on protein-fragment complementation assays , 2007, Nature Reviews Drug Discovery.

[32]  J. Silberg,et al.  A selection that reports on protein-protein interactions within a thermophilic bacterium. , 2010, Protein engineering, design & selection : PEDS.

[33]  S. Ikeda,et al.  Rapid Modification of Proteins Using a Rapamycin-Inducible Tobacco Etch Virus Protease System , 2009, PloS one.

[34]  D. Tirrell,et al.  Chemical Tools for Temporally and Spatially Resolved Mass Spectrometry-Based Proteomics , 2013, Annals of Biomedical Engineering.

[35]  Christopher A. Voigt,et al.  Automated design of synthetic ribosome binding sites to control protein expression , 2016 .

[36]  J. Liao,et al.  Protein engineering for metabolic engineering: Current and next‐generation tools , 2013, Biotechnology journal.

[37]  S. Chandrasegaran,et al.  Protein fragment complementation in M.HhaI DNA methyltransferase. , 2005, Biochemical and biophysical research communications.

[38]  D. Engelman,et al.  TOXCAT: a measure of transmembrane helix association in a biological membrane. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[39]  H. Blau,et al.  Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[40]  P. Schimmel,et al.  Assembly of a class I tRNA synthetase from products of an artificially split gene. , 1991, Biochemistry.

[41]  V. Verkhusha,et al.  Near-infrared fluorescent proteins for multicolor in vivo imaging , 2013, Nature Methods.

[42]  A. Galarneau,et al.  β-Lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein–protein interactions , 2002, Nature Biotechnology.

[43]  R. Hodges,et al.  Designing Heterodimeric Two-stranded α-Helical Coiled-coils , 2002, The Journal of Biological Chemistry.

[44]  Stephanie J. Culler,et al.  In vivo fluorescent detection of Fe-S clusters coordinated by human GRX2. , 2009, Chemistry & biology.

[45]  Michael Z. Lin,et al.  Mammalian Expression of Infrared Fluorescent Proteins Engineered from a Bacterial Phytochrome , 2009, Science.

[46]  H. Piwnica-Worms,et al.  Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. , 2004, Proceedings of the National Academy of Sciences of the United States of America.