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Building blocks for protein interaction devices
- Source :
- Nucleic Acids Research
- Publication Year :
- 2010
- Publisher :
- Oxford University Press (OUP), 2010.
-
Abstract
- Here, we propose a framework for the design of synthetic protein networks from modular protein-protein or protein-peptide interactions and provide a starter toolkit of protein building blocks. Our proof of concept experiments outline a general work flow for part-based protein systems engineering. We streamlined the iterative BioBrick cloning protocol and assembled 25 synthetic multidomain proteins each from seven standardized DNA fragments. A systematic screen revealed two main factors controlling protein expression in Escherichia coli: obstruction of translation initiation by mRNA secondary structure or toxicity of individual domains. Eventually, 13 proteins were purified for further characterization. Starting from well-established biotechnological tools, two general-purpose interaction input and two readout devices were built and characterized in vitro. Constitutive interaction input was achieved with a pair of synthetic leucine zippers. The second interaction was drug-controlled utilizing the rapamycin-induced binding of FRB(T2098L) to FKBP12. The interaction kinetics of both devices were analyzed by surface plasmon resonance. Readout was based on Förster resonance energy transfer between fluorescent proteins and was quantified for various combinations of input and output devices. Our results demonstrate the feasibility of parts-based protein synthetic biology. Additionally, we identify future challenges and limitations of modular design along with approaches to address them.
- Subjects :
- Leucine Zippers
Leucine zipper
business.industry
Recombinant Fusion Proteins
DNA
Tacrolimus Binding Protein 1A
Computational biology
Protein engineering
Surface Plasmon Resonance
Modular design
BioBrick
Biology
Protein Engineering
Synthetic biology
Förster resonance energy transfer
Biochemistry
Proof of concept
Synthetic Biology and Chemistry
Protein Interaction Mapping
Fluorescence Resonance Energy Transfer
Genetics
Protein Interaction Domains and Motifs
Cloning, Molecular
Surface plasmon resonance
business
Subjects
Details
- ISSN :
- 13624962 and 03051048
- Volume :
- 38
- Database :
- OpenAIRE
- Journal :
- Nucleic Acids Research
- Accession number :
- edsair.doi.dedup.....27abd2515a472edb2f240f6f6511537e