Method for protein tagging in Schizosaccharomyces pombe.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Protein tagging is crucial for studying protein localization, function, and expression.
• Rad32 protein in fission yeast (Schizosaccharomyces pombe) is homologous to human Mre11 and plays a role in DNA double-strand break repair and checkpoint control.

Purpose of the Study:

• To N-terminally tag the Rad32 protein in fission yeast using the Cre-loxp recombination system.
• To establish a system for controlled gene expression and subsequent native promoter-driven expression of tagged Rad32.

Main Methods:

• Integration of a DNA fragment containing a tandem affinity purification (TAP) tag, loxP sites, and an inducible promoter (nmt1) upstream of the rad32 gene.
• Utilizing Cre recombinase to excise the tag, marker, and artificial promoter, resulting in the TAP-tagged rad32 gene under its native promoter control.
• Employing Western blotting for protein detection and purification for functional studies.

Main Results:

• Successful integration and excision of the DNA fragment, leading to N-terminally tagged Rad32.
• Establishment of a fission yeast strain expressing tagged Rad32 under its native promoter.
• The tagged strain is prepared for subsequent protein purification and functional characterization.

Conclusions:

• The Cre-loxp system provides an efficient method for tagging Rad32 in fission yeast.
• This tagged Rad32 facilitates future investigations into its role in DNA repair pathways.
• The developed methodology supports protein purification and functional analysis of Rad32.