Abstract
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 the E. coli BirA protein, has been fused to proteins-of-interest, making them substrates for in vivo biotinylation. 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 the Drosophila female 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 secreted Drosophila proteins, 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 BirA in vivo. Our results indicate that secBirA directs biotinylation of proteins bound for secretion in 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.
Highlights
Originating with the pioneering studies of Casadaban, Silhavy, Beckwith and co-workers [1,2,3,4,5], experimental strategies involving the generation of proteins that have been attached genetically to exogenous protein or peptide tags have had an enormous impact upon progress in biological disciplines including biochemistry, cell and developmental biology, genetics, microbiology and molecular biology
In an effort to develop a simple and efficient method for the isolation of secreted proteins relying on the high affinity interaction between avidin and biotin, we initially generated a secreted version of E. coli BirA that was designed to be retained in the endoplasmic reticulum (ER)
Expression of genes cloned into pUASp under the control of the Nanos-Gal4::VP16 driver element [61] leads to the production of protein in the germline-derived ovarian cells (15 nurse cells and oocyte) and in the progeny embryo, respectively, while expression of genes cloned into pUAST under the control of the CY2-Gal4 driver element leads to the production of protein in the ovarian follicle cells [62]
Summary
Originating with the pioneering studies of Casadaban, Silhavy, Beckwith and co-workers [1,2,3,4,5], experimental strategies involving the generation of proteins that have been attached genetically to exogenous protein or peptide tags have had an enormous impact upon progress in biological disciplines including biochemistry, cell and developmental biology, genetics, microbiology and molecular biology. Other protein tags (e.g. Glutathione-S-Transferase, Maltose-Binding Protein) [10,11,12,13,14] have facilitated isolation of proteins-of-interest by affinity chromatography, permitting analyses of their structure, modification, and interaction with other factors. A variety of small peptide tags that interact either with characterized antibodies (several tags comprising characterized epitopes) [15,16,17], Streptavidin/Streptactin (SBP-tag, Strep-tag) [18,19,20], Calmodulin (Calmodulin-tag) [21], Nickel or Cobalt chelate (His-tag) [22,23,24,25,26] or anion exchange resin (polyglutamate tag) [27, 28] have been utilized for the detection or isolation of proteins-of-interest to which they have been fused. Over the course of time, many additional protein tags have been developed with various useful properties [29]
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