A Plasmid System That Utilises Phosphoribosylanthranilate Isomerase to Select Against Cells Expressing Truncated Proteins
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 <i>Saccharomyces cerevisiae</i> from a galactose-inducible promoter. The gene of interest is fused in-frame to a downstream sequence cod...
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| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-03-01
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| Series: | Biomolecules |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2218-273X/15/3/412 |
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| Summary: | 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 <i>Saccharomyces cerevisiae</i> 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. |
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| ISSN: | 2218-273X |