P15 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
Constructs were assembled according to R.F.C. 25. All parts were codon optimized for E. coli.
This is the scar produced by assembly using RFC 25. If you are assembling a composite part using RFC 25, you can insert this part and specify blunt assembly to get the desired sequence.
Strong T7 promoter and RBS including start codon for protein expression in E. coli or in-vitro. Part is provided in RFC 25 assembly format.
* sequence is taken from the commonly used pET3a expression vector * deleted XbaI site between prom. and RBS * T7 promoter sequence (1-19) equals the one in BBa_I712074 * Conservative choice of part boundaries and modifications -- the part's size could probably be reduced.
preScission protease cleavage site (E. coli-optimized)
cleavage site for PreScission??? protease. preScission protease cleavage site (PreScission??? protease is a genetically engineered form of human rhinovirus 3C protease with a GST fusion, allowing for facile cleavage and purification of GST-tagged proteins along with protease removal after recombinant protein cleavage. Enables low-temperature cleavage of fusion proteins containing the eight-residue recognition sequence.) cleavage site: * Leu-Glu-Val-Leu-Phe-Gln↓Gly-Pro References ============== Walker et al. (1994): Efficient and rapid affinity purification of proteins using recombinant fusion proteases. PMID: 7764949
* gene synthesis * codon optimized for E. coli
Highly flexible 10 amino acid linker. Translates to [GS]_5. Codon-optimize for E. coli, yeast, mammalian.
* gene synthesis * codon optimized for E. coli * experimental distance distributions are described in Neuweiler et al. 2006 papers.
The second LOV domain of Arabidopsis phototropin 1 shows light-induced transient (30s) homo-dimerization between a ground state and an excited monomer (Nakasone et al. 2006). LOV2 is predominantly monomeric at concentrations below 100microM but turns dimeric above. Funnily, the high-conc. dimer state experiences photo-dissociation instead. Either the excitation follows a different course from the dimer starting state or the excitation event itself pushes the complex apart. (CRG-internal ID: rg0090) ===References=== * Nakasone et al. 2006 PMID: 16679373 * Freddolino, Peter L.; Dittrich, Markus; Schulten, Klaus (2006) Dynamic Switching Mechanisms in LOV1 and LOV2 Domains of Plant Phototropins PMID: 16935961
The fragment used by Nakasone et al. is 475K - 578G. The core domain 449E - 586R has reportedly lower stability at RT (aggregation). * gene synthesis * codon optimized for E. coli
N-terminal fragment of H. gaussia luciferase for PCA. '''Disulfide bonds:''' * likely, many Cys in sequence ===Purification=== Inouye & Sahara (2008) discuss the purification and in-vitro activity of different Luciferases, including Gaussia. They indicate that Gaussia Luciferase was previously purified from insoluable fractions -- perhaps due to the high Cys content. Goerke et al (2008) Provide optimized in-vitro expression conditions for Gaussia Luciferase based on tuning the strain from which the cell-free lysate is produced. ===References=== http://www.ncbi.nlm.nih.gov/pubmed/17099704 Remy I, Michnick SW. (2006) A highly sensitive protein-protein interaction assay based on Gaussia luciferase. http://nar.oxfordjournals.org/cgi/content/full/27/13/e4#hd1 Maroun M, Aronheim A. (2007) A novel in vivo assay for the analysis of protein-protein interaction. PMID: 10373602 http://www.ncbi.nlm.nih.gov/pubmed/19373229 Tannous BA. (2009) Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. http://www.ncbi.nlm.nih.gov/pubmed/18789309 Inouye S, Sahara Y. (2008) Soluble protein expression in E. coli cells using IgG-binding domain of protein A as a solubilizing partner in the cold induced system. http://www.ncbi.nlm.nih.gov/pubmed/18555198 Goerke AR, Loening AM, Gambhir SS, Swartz JR. (2008) Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase.
Hexahistidine affinity tag. Binds zinc, nickel, or cobalt affinity resins. =Notes= This part is identical to BBa_I757013 (which does not seem to be available though). =References= Wikipedia article: [http://en.wikipedia.org/wiki/Polyhistidine-tag]
Simple repetition of the His codon most frequent in E. coli -- a more balanced codon-choice may be better.
T7 terminator for protein expression in E. coli or in vitro. Part is provided in R.F.C. 25 assembly format.
* taken from pET3a expression plasmid * added two STOP to 5' * conservative choice of part boundary, could probably be shortened
Conversion of the iGEM parts registry to SBOL2.1