Protein splicing in trans by purified N- and C-terminal fragments of the Mycobacterium tuberculosis RecA intein.
Protein splicing involves the self-catalyzed excision of protein splicing elements, or inteins, from flanking polypeptide sequences, or exteins, leading to the formation of new proteins in which the exteins are linked directly by a peptide bond. To study the enzymology of this interesting process we have expressed and purified N- and C-terminal segments of the Mycobacterium tuberculosis RecA intein, each approximately 100 amino acids long, fused to appropriate exteins. These fragments were reconstituted into a functional protein splicing element by renaturation from 6 M urea. When renaturation was carried out in the absence of thiols, the reconstituted splicing element accumulated as an inactive disulfide-linked complex of the two intein fragments, which could be induced to undergo protein splicing by reduction of the disulfide bond. This provided a useful tool for separately investigating the requirements for the reconstitution of the intein fragments to yield a functional protein splicing element and for the protein splicing process per se. For example, the pH dependence of these processes was quite different, with reconstitution being most efficient at pH 8.5 and splicing most rapid at pH 7.0. The availability of such an in vitro protein splicing system opens the way for the exploration of intein structure and the unusual enzymology of protein splicing. In addition, this trans-splicing system is a potential protein ligase that can link any two polypeptides fused to the N- and C-terminal intein segments.
Mills
K V
KV
Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA 02114, USA.
Lew
B M
BM
Jiang
S
S
Paulus
H
H
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008025:Ligases / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D019154:Protein Splicing; D011938:Rec A Recombinases / Q000235:genetics* / Q000378:metabolism; D017421:Sequence Analysis; D013438:Sulfhydryl Compounds
D001426:Bacterial Proteins; D013438:Sulfhydryl Compounds; D011938:Rec A Recombinases; D008025:Ligases