Not just a tool for life scientists: but why is trypsin so useful in the lab?
Along with other serine proteases, physiologists know trypsin as a key enzyme for the digestion of proteins that is both stable and abundant in digestive juices. First described in the late 19th century, trypsin starts out as the proenzyme trypsinogen formed in the pancreas, then delivered via the pancreatic duct for cleavage to its active form in the duodenum. This sequestration of the active enzyme is a safeguard against the indiscriminate, undesirable cleavage of somatic proteins, which can lead to inflammatory pathologies like pancreatitis.
Figure 1. Trypsin is used in multiple biotechnical applications, from detaching adherent cells in culture (left) to digestion of proteins for mass spectrometry. Right, protein from a digestion of E. coli was loaded on an SDS PAGE gel, and a spot was then excised and tryptically digested for MALDI analysis.
Cell culture with adherent phenotypes
Just as it is in digestion, trypsin is a powerfully specific molecular tool that enables precise proteolytic cleavage in the hands of the life scientist. The use of trypsin in cell culture was first reported more than 50 years ago. In cell culture, short incubations with trypsin can cleave proteins to detach adherent cell types from culture surfaces and from each other, permitting the removal of cells from flasks, dishes, and plates—and creating single-cell suspensions for cell counting, passage, and downstream experiments.
Application-specific trypsin: more stability, please
Beyond cell culture, trypsin has other uses in the biological sciences. During proteomics experiments, for example, it is used to digest proteins into peptides for mass spectrometry analysis. Trypsin’s specificity is particularly helpful here, as it breaks down just the peptide bonds in which the carbonyl group is on an arginine or a lysine residue.
Trypsin’s exceptional activity for protein digestion can sometimes present a problem for proteomics research, however. “Without efforts to stabilize it, trypsin will eventually digest itself,” says Tracy Adair-Kirk, Principal Scientist at MilliporeSigma. This is particularly undesirable for applications where autolysis may contaminate and confound experimental results. “Our goal was to stabilize trypsin for mass spec so that we could reduce peaks due to autodigestion fragments,” explains Adair-Kirk. The project started in the hands of Senior Scientist Judy Boland, and resulted in the development of a “pure, recombinant trypsin formulation,” marketed as SOLu-trypsin, an advanced proteomics-grade enzyme. According to Boland, a change to formulation conditions means “SOLu-trypsin doesn’t cleave itself,” so there are no tryptic fragments to contribute to mass spec contaminant peaks—a gift for proteomics research.
Figure 2. Crystal structure of the first active autolysate form of porcine alpha trypsin
Cell culture serendipity: a new thermostable formulation for general detachment
So why develop a different stabilized formulation for general culture applications? “For me, it was easy to make that leap to a thermostable trypsin that didn’t digest itself for cell culture,” recalls Adair-Kirk. While it was necessary for proteomics studies to avoid stabilizing trypsin with additives that might contribute contaminating peaks in mass spec, the goals were different for cell culture. Adair-Kirk, Boland, and team knew from their own experience that the tedium of thawing trypsin stock, aliquoting for single use, refreezing aliquots, and thawing again for subculture meant finding a formulation that was stable outside the freezer, and retained activity at a wide range of lab temperature conditions.
What was surprising is that the new formulation was not only stable at 4°C, but also at ambient temperature, and that it retained >90% activity even when maintained at 37°C. The resulting StableCell™ trypsin is manufactured at three concentrations to allow optimization for a range of cell 'stickiness’. And, unlike other room-temperature detachment reagents, it’s porcine trypsin—so does not require validation in culture systems already using traditional trypsin formulations.
Let your application be your guide: Why trypsin formulations are not all the same
Many labs may purchase the most common formulation of trypsin regardless of application—often 0.25% trypsin plus ethylenediaminetetraacetic acid (EDTA), augmented with phenol red as a pH indicator. Though typically not robust enough to detach most adherent cells on its own, EDTA provides a synergistic mechanism for dissociating cells when added to trypsin: it pulls calcium out of cell-surface and cell-cell bonds, chelates free Ca2+ in the media that can inhibit trypsin activity, and enables the use of lower concentrations of trypsin, which can be harsh to cells when concentration is high or incubation is too long. Phenol red is also frequently included as a monitor of pH, as trypsin activity is optimal in the 7-9 pH range. However, formulations without EDTA or phenol red are often available for applications where they might be contraindicated.
Figure 3. Activity of standard vs stabilized trypsin over time
Tips for choosing—and using—trypsin formulations for life science applications
To ‘round up’, below are some tips for choosing the right trypsin formulation:
Like to try StableCell™ Trypsin in your culture room? Vist our product page for more details.