resources

PROTEIN DESIGN methods

RosettaScripts protocols are available on GitHub.


wet lab methods

laboratory protocols are available on GitHub


expression vectors

plasmids are available from addgene and MolecularCloud


These vectors are optimized for gene insertion by Gibson assembly, and all proteins will be purified using immobilized-metal affinity chromatography.


vectors for standard protein expression

  • pCDB736: N-terminal 10-His SUMO AviTag 5xGGS TEV-peptide fusion
    • the AviTag enables enzymatic ligation of biotin to the protein
    • scarless removal of the 10-His SUMO tag is accomplished using SUMO protease, leaving the AviTag attached to the protein N-terminus by a long, flexible linker
    • scarless removal of the entire fusion domain is accomplished using TEV protease
  • pCDB24 or pCDB179: N-terminal 10-His SUMO fusion
  • pCDB347: N-terminal 10-His TEV-peptide fusion
      • scarless removal of the His tag is accomplished using TEV protease
  • pCDB97: N-terminal 6-His fusion
      • this His tag is not cleavable
  • pCDB99 or pCDB348: C-terminal 6-His fusion
      • this His tag is not cleavable
  • pCDB256: N-terminal 10-His fusion
      • this His tag is not cleavable
  • pCDB98: C-terminal 10-His fusion
      • this His tag is not cleavable

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