PDF《protein》

30 nucleotides;

however, unlike most other homing endonucleases, it acts as a monomer on its recognition sites [9, 10]. To study proteins in-vivo, green fluorescence protein (GFP) provides an excellent means for monitoring gene expression and protein localization in cells. GFP was first isolated from the jellyfish Aequorea in the early 1960'

s [11], however, identification of its sequence and its first use as a reporter gene didn'

t occur for another

30 years [12, 13]. Since then GFP has been used frequently throughout the biological sciences. Typically, the GFP marker is attached to the carboxyl or amino terminus of proteins. However, introducing the GFP inside another protein might not significantly distort GFP'

s structure. This is because, as shown in figure 1A, both termini of GFP appear at one end of the folded protein, and are rather flexible on the surface of the so-called β-can [14]. The PI-SceI homing endonuclease structure contains numerous loops which are distant from the active sites;

suggesting that it might be possible to insert a functional GFP without disrupting either the self-splicing or the endonuclease activities. Here we describe insertion of a GFP domain within a loop of the Int. J. Biol. Sci. 2007,

3 206 sub-domain of the Sce VMA1 intein. The new recombinant protein was visualized using fluorescent microscopy indicating functional GFP;

the tagged intein excises from the host protein;

and the new GFP tagged homing endonuclease was functional. However, distinct from the wild type PI-SceI homing endonuclease, the new protein cleaves its target site only in presence of Mn2+. 2. Materials and Methods PI-SceI cloning in pET expression plasmid Plasmids containing the Saccharomyces cerevisiae V-ATPase catalytic subunit gene (vma1), with and without intein, [15] were kindly provided by Dr. Frederick S. Gimble (Purdue University). The gene encoding V-ATPase A-subunit intein was amplified by PCR using primers VMA-1 and VMA-2 (see Table 1). Following digestion with NdeI and BamHI, the coding sequence was cloned to the vector pET-15b (Novagen), behind the T7 promoter, for g........