Work

A Plasmid System That Utilises Phosphoribosylanthranilate Isomerase to Select Against Cells Expressing Truncated Proteins

Year: 2025

Type: article

Abstract: We have generated a vector that enables the removal of plasmids coding for truncated proteins. This vector expresses a protein of interest in the yeast Saccharomyces cerevisiae from a galactose-inducible promoter. The gene of interest is fused in-frame to a downstream sequence coding for phosphoribosylanthranilate isomerase (PRAI), which catalyses the third step in tryptophan biosynthesis. As a consequence, only the full-length protein of interest renders the host cell tryptophan prototrophic, allowing for selection against cells expressing truncated proteins. Our proof-of-principle study demonstrates that PRAI is functional when fused C-terminally to a protein, robustly rendering cells tryptophan prototrophic. The N-terminal GST tag and C-terminal myc tag allow for tag-mediated protein purification, co-precipitation studies, determination of relative expression levels, as well as validation of full-length expression of the protein via Western blotting.

Source: Biomolecules

DOI: 10.3390/biom15030412

Authors: Aditi A. Ghuge, Susanne Gottfried, Anja H. Schiemann, Evelyn Sattlegger

Institutions: Massey University, Maurice Wilkins Centre

Language: English

Topic: Fungal and yeast genetics research

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,258

Funder: Massey University