Saporin is a ribosome-inactivating protein that can cause inhibition of protein synthesis and causes cell death when delivered inside a cell. Development of commercial Saporin results in a technology termed ‘molecular surgery’, with Saporin as the scalpel. Its low toxicity (it has no efficient method of cell entry) and sturdy structure make Saporin a safe and simple molecule for many purposes. The most popular applications use experimental molecules that deliver Saporin via an add-on targeting molecule. These add-ons come in several forms: peptides, protein ligands, antibodies, even DNA fragments that mimic cell-binding ligands. Cells that do not express the targeted cell surface marker will not be affected. This review will highlight some newer efforts and discuss significant and unexpected impacts on science that molecular surgery has yielded over the last almost four decades. There are remarkable changes in fields such as the Neurosciences with models for Alzheimer’s Disease and epilepsy, and game-changing effects in the study of pain and itch. Many other uses are also discussed to record the wide-reaching impact of Saporin in research and drug development.

Streptavidin-Saporin can be considered a type of ‘secondary’ targeted toxin. The scientific community has taken advantage of this conjugate in clever and fruitful ways using many kinds of biotinylated targeting agents to send saporin into a cell selected for elimination. Saporin is a ribosome-inactivating protein that causes inhibition of protein synthesis and cell death when delivered inside a cell. Streptavidin-Saporin, mixed with biotinylated molecules to cell surface markers, results in powerful conjugates that are used both in vitro and in vivo for behavior and disease research. Streptavidin-Saporin harnesses the ‘Molecular Surgery’ capability of saporin, creating a modular arsenal of targeted toxins used in applications ranging from the screening of potential therapeutics to behavioral studies and animal models. The reagent has become a well-published and validated resource in academia and industry. The ease of use and diverse functionality of Streptavidin-Saporin continues to have a significant impact on the life science industry.

Quick and efficient screening of targeting agents that internalize effectively is vital for determining their suitability as potential therapeutics. Some of the most recent successes in the treatment of cancers have been from antibodies to cell surface proteins that are responsible for tumor cell proliferation. Examples are Cetuximab (target: EGFR) approved for colorectal cancer, and Trastuzumab (target: HER2) for breast cancer. These antibodies have more than one effect on the cancer cell, but one of the most important is that, upon binding to the cell surface antigen, the complex is internalized. As such, the down-regulated cell surface protein no longer plays a role in cancer cell division. Here we describe a method for determining internalization of cell surface molecules by targeting agents using a pH-dependent fluorescent reporter cross-linked to streptavidin. Streptavidin is a tetrameric protein (molecular weight 53 kDa in its recombinant form), with each subunit able to bind a single biotin molecule. The bond between streptavidin and biotin is rapid and essentially non-reversible, unaffected by most extremes of pH, organic solvents, and denaturing reagents. It is the strongest known noncovalent biological interaction (Ka = 1015 M-1) between protein and ligand. A variety of molecules, including lectins, proteins, and antibodies, can be biotinylated and reacted with streptavidin-labeled probes or other detection reagents for use in biological assays. The fluorescence from this reporter increases intensity as the pH of its surroundings becomes more acidic, as demonstrated when exposed to the environment inside a cell (thereby providing evidence of internalization). Here we describe methods that can be used to explore candidates as cancer therapeutics in a quick, reliable and reproducible manner. Citation Format: Patrick A. Shramm, Leonardo Ancheta. Streptavidin-pHast: A readily conjugatable, pH-sensitive dye to screen for internalization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2177.