Controllable protein cleavages through intein fragment complementation.
Intein-based protein cleavages, if carried out in a controllable way, can be useful tools of recombinant protein purification, ligation, and cyclization. However, existing methods using contiguous inteins were often complicated by spontaneous cleavages, which could severely reduce the yield of the desired protein product. Here we demonstrate a new method of controllable cleavages without any spontaneous cleavage, using an artificial S1 split-intein consisting of an 11-aa N-intein (I(N)) and a 144-aa C-intein (I(C)). In a C-cleavage design, the I(C) sequence was embedded in a recombinant precursor protein, and the small I(N) was used as a synthetic peptide to trigger a cleavage at the C-terminus of I(C). In an N-cleavage design, the short I(N) sequence was embedded in a recombinant precursor protein, and the separately produced I(C) protein was used to catalyze a cleavage at the N-terminus of I(N). These N- and C-cleavages showed >95% efficiency, and both successfully avoided any spontaneous cleavage during expression and purification of the precursor proteins. The N-cleavage design also revealed an unexpected and interesting structural flexibility of the I(C) protein. These findings significantly expand the effectiveness of intein-based protein cleavages, and they also reveal important insights of intein structural flexibility and fragment complementation.
Volkmann
Gerrit
G
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
Sun
Wenchang
W
Liu
Xiang-Qin
XQ
D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D047668:Inteins / Q000235:genetics*; D007700:Kinetics; D058619:Maltose-Binding Proteins; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods*; D019154:Protein Splicing / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013879:Thioredoxins