The protocol presents a method for in vivo covalent attachment of biotin to proteins based on their proximity to a biotin ligase fused to a protein of interest. This modification allows for a selective enrichment of the proteins using streptavidin beads as needed in protein interaction studies.

Paramecium is a unicellular eukaryote with nuclear dimorphism. In its germline nucleus, many protein coding genes are interrupted by transposon-like sequences that must be removed in the somatic nucleus. This requires multi-protein complexes in a dynamic process.

One efficient strategy to study such intricate molecular machinery is by proximity biotinylation. In our approach, we can insert any target gene upstream of turboID ligase to create a fusion protein. Then this construct needs to be injected into the somatic nucleus of paramecium, as demonstrated in the following protocol.

For this protocol used today, cells that have been recovered in fresh culture medium. Use a stereo microscope to capture the cells with the pipette and transfer them to a fresh depression slide field with washing medium. While the cells are in the washing medium, disperse 20 microliters of water on the glass slide and overlay with the cover glass.

Add 200 microliters of mineral oil on top of the cover glass. After this, transfer individual cells from the washing slide as droplets underneath the mineral oil. Position the prepared slide on the pedestal of the injection microscope.

Attach the aspiration needle and move it into position. Activate the robotic arm and the FemtoJet microinjector. Use a microloader pipette tip to add the DNA inside the capillary of the injection needle.

Attach the injection needle to the injection system, then move it into position, and connect to the FemtoJet. Use the robotic arm to position the injection needle inside a water droplet. Then press clean in order to purge the needle until flow of DNA is visible.

Move on to a droplet containing a cell, and lower the aspiration needle. Then gently aspirate the water until the cell is immobilized. Raise the aspiration needle and lower the injection needle.

The injection needle needs to be inserted into a darker area corresponding to the somatic nucleus. A diffuse droplet may become visible within the boundaries of the somatic nucleus upon a successful injection. After the injection, use the aspiration needle to return the water to the cell.

Next, each injected cell needs to be recovered into a well filled with culture medium. After expanding the cells, the presence of the injected DNA needs to be validated. A success rate of 40 to 50%is usually expected with this procedure.

It is also necessary to validate the successful expression of proteins from the injected DNA as well as their correct localization using immunofluorescent staining. Here, we show the successful use of a NOVA1 protein fused to a turboID biotin ligase for proximity labeling during different developmental stages in paramecium. Notably, supplemental biotin increases the biotinylation in cells which express a turboID fusion protein.

However, the addition of biotin to wild-type cells does not lead to biotinylation. As expected, the biotin is covalently attached to various proteins, as shown in the Western blot analysis. Finally, we use streptavidin coated magnetic beads to demonstrate that the biotinylated proteins can be selectively enriched from a paramecium lysate.

We developed a method for biotin tagging of proteins in vivo in paramecium. This method is rapidly adaptable to any gene of interest. Stage specific biotinylation can be achieved through timing of the biotin supplementation.