Isolation of secreted proteins from Drosophila ovaries and embryos through in vivo BirA-mediated biotinylation
| Autor: | Yuri Volnov, David Stein, Yuan Zhang, Leslie M. Stevens, Geng Chen |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
B Vitamins
Embryology Embryo Nonmammalian Protein Extraction Endoplasmic Reticulum Biochemistry chemistry.chemical_compound 0302 clinical medicine Biotin Protein purification Medicine and Health Sciences Drosophila Proteins Carbon-Nitrogen Ligases Post-Translational Modification Extraction Techniques 0303 health sciences Secretory Pathway Multidisciplinary biology Organic Compounds Chemistry Drosophila Melanogaster Escherichia coli Proteins Eukaryota Animal Models Vitamins Cell biology Ovaries Insects Experimental Organism Systems Cell Processes Biotinylation Physical Sciences Medicine Drosophila Female Anatomy Cellular Structures and Organelles Drosophila Protein Research Article animal structures Arthropoda Science Research and Analysis Methods 03 medical and health sciences Model Organisms In vivo Escherichia coli Animals Secretion Protein Interactions 030304 developmental biology Endoplasmic reticulum Embryos Organic Chemistry Ovary Reproductive System Organisms Chemical Compounds Biology and Life Sciences Proteins Cell Biology Invertebrates Repressor Proteins Secretory protein Animal Studies biology.protein 030217 neurology & neurosurgery Developmental Biology Avidin |
| Zdroj: | PLoS ONE PLoS ONE, Vol 14, Iss 10, p e0219878 (2019) |
| DOI: | 10.1101/694091 |
| Popis: | The extraordinarily strong non-covalent interaction between biotin and avidin (kD = 10-14-10-16) has permitted this interaction to be used in a wide variety of experimental contexts. The Biotin Acceptor Peptide (BAP), a 15 amino acid motif that can be biotinylated by theE. coliBirA protein, has been fused to proteins of interest, making them substrates forin vivobiotinylation. Here we report on the construction and characterization of a modified BirA bearing signals for secretion and endoplasmic reticulum (ER) retention, for use in experimental contexts requiring biotinylation of secreted proteins. When expressed in theDrosophilafemale germline or ovarian follicle cells under Gal4-mediated transcriptional control, the modified BirA protein could be detected and shown to be enzymatically active in ovaries and progeny embryos. Surprisingly, however, it was not efficiently retained in the ER, and instead appeared to be secreted. To determine whether this secreted protein, now designated secBirA, could biotinylate secreted proteins, we generated BAP-tagged versions of two secretedDrosophilaproteins, Torsolike (Tsl) and Gastrulation Defective (GD), which are normally expressed maternally and participate in embryonic pattern formation. Both Tsl-BAP and GD-BAP were shown to exhibit normal patterning activity. Co-expression of Tsl-BAP together with secBirA in ovarian follicle cells resulted in its biotinylation, which permitted its isolation from both ovaries and progeny embryos using Avidin-coupled affinity matrix. In contrast, co-expression with secBirA in the female germline did not result in detectable biotinylation of GD-BAP, possibly because the C-terminal location of the BAP tag made it inaccessible to BirAin vivo. Our results indicate that secBirA directs biotinylation of proteins bound for secretionin vivo, providing access to powerful experimental approaches for secreted proteins of interest. However, efficient biotinylation of target proteins may vary depending upon the location of the BAP tag or other structural features of the protein. |
| Databáze: | OpenAIRE |
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