Nucleic Acids Research, Vol 26, Issue 22 5109-5115, Copyright © 1998 by Oxford University Press
S Chong, GE Montello, A Zhang, EJ Cantor, W Liao, MQ Xu and J Benner
A conventional affinity protein purification system often requires a
separate protease to separate the target protein from the affinity tag.
This paper describes a unique protein purification system in which the
target protein is fused to the C-terminus of a modified protein splicing
element (intein). A small affinity tag is inserted in a loop region of the
endonuclease domain of the intein to allow affinity purification. Specific
mutations at the C-terminal splice junction of the intein allow
controllable C-terminal peptide bond cleavage. The cleavage is triggered by
addition of thiols such as dithiothreitol or free cysteine, resulting in
elution of the target protein while the affinity-tagged intein remains
immobilized on the affinity column. This system eliminates the need for a
separate protease and allows purification of a target protein without the
N-terminal methionine. We have constructed general cloning vectors and
demonstrated single-column purification of several proteins. In addition,
we discuss several factors that may affect the C-terminal peptide bond
cleavage activity.
ARTICLES
Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step
New England Biolabs Inc., 32 Tozer Road, Beverly, MA 01915, USA. chong@neb.com
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