Protein splicing in vitro with a semisynthetic two-component minimal intein.
Protein splicing elements, or inteins, catalyze their own excision from flanking polypeptide sequences, or exteins, thereby leading to the formation of new proteins in which the exteins are linked directly by a peptide bond. A trans-splicing system, using separately purified and expressed N- and C-terminal intein fragments of about 100 amino acids each, fused to appropriate exteins, was recently derived from the Mycobacterium tuberculosis RecA intein (Mills, K. V., Lew, B. M., Jiang, S.-Q., and Paulus, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3543-3548). We have replaced the C-terminal intein fragment of this system with synthetic peptides comprising 35-50 of the C-terminal residues of the RecA intein. The N-terminal intein fragment and the synthetic peptide were reconstituted by renaturation from guanidinium chloride. In the absence of added reductants, a disulfide-linked dimer of the N-terminal fragment and the peptide accumulated and could be induced to splice by reduction of its disulfide bond. The intermediate and spliced products were identified by polyacrylamide gel electrophoresis, mass spectrometry, and derivatization with thiol-reactive biotin followed by Western blotting with a streptavidin-enzyme conjugate. This is the first example of protein splicing involving a synthetic intein fragment and opens the way for studying the active site structure and function of the intein by the use of different synthetic peptides, including ones with non-natural amino acids.
Lew
B M
BM
Boston Biomedical Research Institute, Boston, Massachusetts 02114, USA.
Mills
K V
KV
Paulus
H
H
D000595:Amino Acid Sequence; D002384:Catalysis; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D019154:Protein Splicing; D011938:Rec A Recombinases / Q000378:metabolism; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010455:Peptides; D011938:Rec A Recombinases