ORFtag: innovative method to tag and study proteins in cells

Studying the function of proteins is key to understanding cellular processes in health and disease. Scientists have long used genetic methods for this type of research; however, these had several limitations, from the scarcity of cell models to costs. In a collaborative effort drawing from scientists across the Vienna BioCenter, researchers from the Research Institute of Molecular Pathology (IMP), the Institute of Molecular Biotechnology (IMBA), and the Max Perutz Labs now developed a method that allows the systematic study of protein function by mass tagging and testing their activity in cells. The study was published in Nature Methods.

Proteins play vital roles in every cellular process. Understanding the molecular function of each protein and how they interact with one another is a crucial objective in modern biology.

Classic approaches like genetic loss-of-function and gain-of-function methods—where gene activity is eliminated or stimulated—have long been employed for this purpose. However, these methods have limitations: in loss-of-function experiments, scientists deactivate a gene to observe the resulting effects, providing insights into the processes a particular protein is involved in. However, this is only possible in some cell models, and is limited by the essentiality of many genes. Conversely, gain-of-function strategies involve activating a gene to study its function, or guide proteins to specific sites in the cell where their activity can be analysed. Such approaches, however, rely on extensive collections of genetic blueprints, known as ‘open reading frames’ (ORFs), which are costly and therefore inaccessible to many laboratories.

In a joint collaborative effort, researchers from the labs of Alexander Stark at the IMP, Stefan Ameres at the Max Perutz Labs, as well as Julius Brennecke and Ullrich Elling at IMBA, have now developed ORFtag–a powerful, affordable, and easy to use method for studying proteins, enabling a comprehensive understanding of their functions. The scientists, led by two students in the Vienna BioCenter PhD Program, Filip Nemcko (IMP) and Moritz Himmelsbach (Max Perutz Labs), published the method in the current issue of the journal Nature Methods.

By leveraging a retrovirus—a type of virus that can sneak its genetic material into the DNA of the cells it infects—ORFtag inserts a small piece of DNA at millions of random positions in the target cell's genome. This DNA produces a "tag" that attaches to newly made proteins, making it possible to identify and study them in large-scale parallel assays. ORFtag enables researchers to explore the roles of nearly all proteins in a cell efficiently and affordably, opening up new possibilities for studying cellular functions and disease mechanisms.