Method for protein tagging in Schizosaccharomyces pombe.
Tagging is a useful method for the investigation of proteins. It allows the localization of the proteins in the cell, their purification in order to investigate their function and the determination of their expression. The aim of the present study was to tag the Rad32 protein of fission yeast (which is the homologue of Mre11 protein from humans) at its N-terminus. Rad32p as well as Mre11p are involved in the repair of DNA double strand breaks and in the DNA damage checkpoint. We carried out this tagging using the Cre-loxp recombination system. In a first step, a 2 kb DNA fragment was integrated upstream of the initiating codon of rad32 gene. This fragment encoded the TAP-tag (tandem affinity purification), a loxp site, a selectable marker (sup3-5), an exogenous promoter (nmt1) and a second loxp site, in this sequence. Following transformation of this DNA fragment into S. pombe cells, rad32 was under the control of the artificial promotor, which allows a controlled expression of the gene by thiamine. In a second step, the cells were transformed with a plasmid coding for Cre recombinase, which catalyses the excision of the DNA sequence between the two loxp sites, removing the marker and the artificial promotor. Thus the tag became attached to the rad32 gene upstream of the ATG, placing the gene under the control of its native promotor. The strain thus obtained will be subsequently used for evidencing the tagged protein by Western blotting and then for its purification in order to investigate its function.
Petrescu-Dănilă
Elena
E
Gr. T. Popa University of Medicine and Pharmacy Iaşi, School of Medicine, Biochemesttry Department.
Voicu
Pia-Manuela
PM
Poiţelea
M
M
Stoica
B
B
Stănescu
Raluca
R
Rusu
M
M
D015153:Blotting, Western; D004706:Endodeoxyribonucleases / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000235:genetics; D020224:Expressed Sequence Tags; D006801:Humans; D019426:Integrases / Q000235:genetics; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics*