Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation.
The determinants governing the self-catalyzed splicing and cleavage events by a mini-intein of 154 amino acids, derived from the dnaB gene of Synechocystis sp. were investigated. The residues at the splice junctions have a profound effect on splicing and peptide bond cleavage at either the N- or C-terminus of the intein. Mutation of the native Gly residue preceding the intein blocked splicing and cleavage at the N-terminal splice junction, while substitution of the intein C-terminal Asn154 resulted in the modulation of N-terminal cleavage activity. Controlled cleavage at the C-terminal splice junction involving cyclization of Asn154 was achieved by substitution of the intein N-terminal cysteine residue with alanine and mutation of the native C-extein residues. The C-terminal cleavage reaction was found to be pH-dependent, with an optimum between pH6.0 and 7.5. These findings allowed the development of single junction cleavage vectors for the facile production of proteins as well as protein building blocks with complementary reactive groups. A protein sequence was fused to either the N-terminus or C-terminus of the intein, which was fused to a chitin binding domain. The N-terminal cleavage reaction was induced by 2-mercaptoethanesulfonic acid and released the 43kDa maltose binding protein with an active C-terminal thioester. The 58kDa T4 DNA ligase possessing an N-terminal cysteine was generated by a C-terminal cleavage reaction induced by pH and temperature shifts. The intein-generated proteins were joined together through a native peptide bond. This intein-mediated protein ligation approach opens up novel routes in protein engineering.
Mathys
S
S
New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915, USA.
Evans
T C
TC
Chute
I C
IC
Wu
H
H
Chong
S
S
Benner
J
J
Liu
X Q
XQ
Xu
M Q
MQ
D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D000458:Cyanobacteria / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D053488:DnaB Helicases; D006868:Hydrolysis; D009154:Mutation; D012326:RNA Splicing
D001426:Bacterial Proteins; D017931:DNA Primers; D001216:Asparagine; D004265:DNA Helicases; D053488:DnaB Helicases; D003545:Cysteine