Characteristics of protein splicing in trans mediated by a semisynthetic split intein.
Protein splicing in trans results in the ligation of two protein or peptide segments linked to appropriate intein fragments. We have characterized the trans-splicing reaction mediated by a naturally expressed, approximately 100-residue N-terminal fragment of the Mycobacterium tuberculosis intein and a synthetic peptide containing the 38 C-terminal intein residues, and found that the splicing reaction was very versatile and robust. The efficiency of splicing was nearly independent of temperature between 4 and 37 degrees C and pH between 6.0 and 7.5, with only a slight decline at pH values as high as 8.5. In addition, there was considerable flexibility in the choice of the C-terminal intein fragment, no significant difference in protein ligation efficiency being observed between reactions utilizing the N-terminal fragment and either the naturally expressed 107-residue C-terminal portion of the intein, much smaller synthetic peptides, or the 107-residue C-terminal intein fragment modified by fusion of a maltose binding protein domain to its N-terminus. The ability to use different types of the C-terminal intein fragments and a broad range of reaction conditions make protein splicing in trans a versatile tool for protein ligation.
Lew
B M
BM
Boston Biomedical Research Institute, Boston, MA 02114, USA.
Mills
K V
KV
Paulus
H
H
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000138:chemical synthesis*; D015202:Protein Engineering / Q000379:methods / Q000639:trends; D019154:Protein Splicing; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000138:chemical synthesis; D013997:Time Factors
D010446:Peptide Fragments; D011994:Recombinant Proteins