Expression of N-terminal Cys-protein fragments using an intein refolding strategy

Bioorganic and Medicinal Chemistry

Volumn 14, Issue 14, 2006, Pages 5043-5048

  Hackenberger, Christian P R ^a   Chen, Mark M ^a   Imperiali, Barbara ^a  

^a MASSACHUSETTS INSTITUTE OF TECHNOLOGY   (United States)

Author keywords

Intein expression; N terminal Cys proteins; Native chemical ligation; Protein semisynthesis

Indexed keywords

CYSTEINE; HYBRID PROTEIN; POLYPEPTIDE; THIOESTER;

AMINO TERMINAL SEQUENCE; ARTICLE; PH; PROTEIN EXPRESSION; PROTEIN PURIFICATION; WESTERN BLOTTING;

BACTERIAL PROTEINS; CARRIER PROTEINS; CLONING, MOLECULAR; CYSTEINE; DNA HELICASES; DNAB HELICASES; ESCHERICHIA COLI PROTEINS; HYDROGEN-ION CONCENTRATION; INCLUSION BODIES; INTEINS; PEPTIDE FRAGMENTS; PROTEIN FOLDING; RECOMBINANT FUSION PROTEINS; SYNECHOCYSTIS;

EID: 33646810065     PISSN: 09680896     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmc.2006.03.003     Document Type: Article

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27. During our experiments we found that stepwise refolding was necessary to re-establish intein functionality. Neither loading the fusion protein under mild denaturation conditions (4 M urea, compare Ref. 15b) followed by removal of urea nor dialysis of the denatured fusion protein against a buffer containing no urea (compare Ref. 4) delivered material capable of intein cleavage.
28. In a separate experiment, a non-reducing peptide gel was performed after on-column cleavage at pH 6.0. This gel showed two peptide bands, which correlate with the desired Ala29Cys-87 peptide and the oxidized disulfide dimer (data not shown).