Expression of N-terminal Cys-protein fragments using an intein refolding strategy.
The inclusion body expression and refolding of a pH-sensitive intein fusion protein (Ssp DnaB intein) delivered sufficient quantities of an N-terminal Cys-polypeptide for native chemical ligations. This strategy circumvents premature intein cleavage under expression conditions and allows the expression and purification of proteins with uncertain solubility properties. The expressed protein resembles the C-terminal portion of the amphiphilic immunity protein Im7, which can be ligated to synthetic thioesters to yield synthetic protein analogues for protein folding studies.
Massachusetts Institute of Technology, Department of Chemistry and Department of Biology, 77 Massachusetts Ave. 18-590, Cambridge, MA 02139, USA.
Christian P R
CP
Hackenberger
Mark M
MM
Chen
Barbara
B
Imperiali
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053488:DnaB Helicases; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000378:metabolism; D047668:Inteins; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046939:Synechocystis / Q000235:genetics
D001426:Bacterial Proteins; D002352:Carrier Proteins; C509253:E colicin-binding immunity protein Im7, E coli; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D004265:DNA Helicases; D053488:DnaB Helicases; D003545:Cysteine