Semisynthesis of cytotoxic proteins using a modified protein splicing element.
Two cytotoxic proteins, bovine pancreatic ribonuclease A (RNase A), and a restriction endonuclease from Haemophilus parainfluenzae (HpaI), were produced using a novel semisynthetic approach that utilizes a protein splicing element, an intein, to generate a reactive thioester at the C-terminus of a recombinant protein. Nucleophilic attack on this thioester by the N-terminal cysteine of a synthetic peptide ultimately leads to the ligation of the two reactants through a native peptide bond. This strategy was used to produce RNase A and HpaI by isolating inactive truncated forms of these proteins, the first 109 and 223 amino acids of RNase A and HpaI, respectively, as fusion proteins consisting of the target protein, an intein, and a chitin binding domain. Thiol-induced cleavage of the precursor led to the liberation of the target protein with a C-terminal thioester-tag. Addition of synthetic peptides representing the amino acids missing from the truncated forms led to the generation of full-length products that displayed catalytic activity indicative of the wild-type enzymes. The turnover numbers and Km for ligated and renatured RNase A were 8.2 s(-1) and 1.5 mM, in good agreement with reported values of 8.3 s(-1) and 1.2 mM (Hodges & Merrifield, 1975). Ligated HpaI had a specific activity of 0.5-1.5 x 10(6) U/mg, which compared favorably with the expected value of 1-2 x 10(6) U/mg (J. Benner, unpubl. obs.). Besides assisting in the production of cytotoxic proteins, this technique could allow the easy insertion of unnatural amino acids into a protein sequence.
Evans
T C
TC
New England Biolabs Inc., Beverly, Massachusetts 01915, USA.
Benner
J
J
Xu
M Q
MQ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002686:Chitin / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004951:Esterification; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins; D012259:Ribonuclease, Pancreatic / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D013438:Sulfhydryl Compounds / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D002686:Chitin; C024406:endodeoxyribonuclease HpaI; D015252:Deoxyribonucleases, Type II Site-Specific; D012259:Ribonuclease, Pancreatic