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J Biol Chem, Vol. 274, Issue 7, 3923-3926, February 12, 1999
From New England Biolabs, Inc.,
Beverly, Massachusetts 01915-5599
The smallest known intein, found in the
ribonucleoside diphosphate reductase gene of Methanobacterium
thermoautotrophicum (Mth RIR1 intein), was found to
splice poorly in Escherichia coli with the naturally
occurring proline residue adjacent to the N-terminal cysteine of the
intein. Splicing proficiency increased when this proline was replaced
with an alanine residue. However, constructs that displayed efficient
N- and C-terminal cleavage were created by replacing either the
C-terminal asparagine or N-terminal cysteine of the intein,
respectively, with an alanine. Furthermore, these constructs were used
to specifically generate complementary reactive groups on protein
sequences for use in ligation reactions. Reaction between an
intein-generated C-terminal thioester on E. coli
maltose-binding protein (43 kDa) and an intein-generated cysteine at
the N terminus of either T4 DNA ligase (56 kDa) or thioredoxin (12 kDa)
resulted in the ligation of the proteins through a native peptide bond. Thus the smallest of the known inteins is capable of splicing and its
unique properties extend the utility of intein-mediated protein
ligation to include the in vitro fusion of large,
bacterially expressed proteins.
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