computational methods

RosettaScripts protocols are available on GitHub.

wet lab methods

laboratory protocols are available on GitHub

bacterial expression vectors

plasmids are available from addgene

These vectors are optimized for gene insertion by Gibson assembly, and all proteins will be purified using immobilized-metal affinity chromatography. All vectors either carry a gene for β-lactamase or aminoglycoside phosphotransferase, granting resistance to ampicillin or kanamycin to bacteria carrying the plasmid. All vectors with ampicillin resistance also carry a yeast origin of replication and uracil synthesis cassette for selection using Ura-dropout media, enabling the vector to be used with Saccharomyces cerevisiae; this allows for cloning using yeast recombineering methods.

vectors for standard protein expression

vectors for expression of proteins with one or more disulfide bonds

  • pCDB26 or pCDB180: N-terminal OsmY (for secretion) 10-His SUMO fusion
      • scar-less removal of the His tag is accomplished using SUMO protease
  • pCDB28 or pCDB380: N-terminal OsmY (for secretion) 10-His TEV-peptide fusion
  • pCDB364: N-terminal 10-His DsbC TEV-peptide fusion
      • this is a fusion to the mature form (i.e. the secretion signal peptide has been removed) of E. coli disulfide bond isomerase, which promotes expression and folding of proteins containing multiple disulfide bonds from the cytoplasm
      • scarless removal of the fusion domain is accomplished using TEV protease
  • pCDB424: N-terminal 10-His DsbC SUMO fusion
      • this is a fusion to the mature form of E. coli disulfide bond isomerase
      • scarless removal of the fusion domain is accomplished using SUMO protease
  • pCDB435: N-terminal 10-His DsbC TEV-peptide AviTag fusion
      • the AviTag enables enzymatic ligation of biotin to the protein, and this will remain attached to the inserted gene following digestion with protease and removal of the fusion domain
  • pCDB441: N-terminal 10-His DsbC SUMO AviTag fusion

miscellaneous expression tools

  • pCDB129: N-terminal 10-His mTurquoise2 and C-terminal SYFP2
      • the inserted gene will be expressed as a fusion between a FRET pair of fluorescent proteins
  • pCDB363: N-terminal pelB signal peptide (for secretion), and a C-terminal 6-His AviTag