Creation of an artificial bifunctional intein by grafting a homing endonuclease into a mini-intein.
The majority of inteins are comprised of a protein splicing domain and a homing endonuclease domain. Experimental evidence has demonstrated that the splicing domain and the endonuclease domain in a bifunctional intein are largely independent of each other with respect to both structure and activity. Here, an artificial bifunctional intein has been created through the insertion of an existing homing endonuclease into a mini-intein that is naturally lacking this functionality. The gene for I-CreI, an intron-encoded homing endonuclease, was grafted into the monofunctional Mycobacterium xenopi GyrA intein at the putative site of the missing endonuclease. The resulting fusion protein was found to be capable of protein splicing similar to that of the parent intein. In addition, the protein demonstrated site-specific endonuclease activity that is characteristic of the I-CreI homing endonuclease. The function of each domain therefore remained unaffected by the presence of the other domain. This artificial fusion of the two domains is a potential novel mobile genetic element.
Fitzsimons Hall
Marilena
M
New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915, USA.
Noren
Christopher J
CJ
Perler
Francine B
FB
Schildkraut
Ira
I
D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000235:genetics; D027081:DNA Gyrase / Q000235:genetics* / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000235:genetics* / Q000378:metabolism; D019912:Mycobacterium xenopi / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D019154:Protein Splicing; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D020418:Algal Proteins; D001426:Bacterial Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI; D027081:DNA Gyrase