The classic structural biology pipeline begins with hypothesizing where protein domains might be found encoded in a larger gene, followed by amplifying that gene fragment by PCR, putting it into a plasmid, expressing it in E. coli, and divining whether the resultant protein fragment is soluble.
“The problem is that a lot of the time this just doesn’t work—despite your best guess the protein fragment is insoluble,” said Darren Hart, Ph.D., team leader at the EMBL in Grenoble, France. “So you have to go ‘round the cycle and re-hypothesize what might be the best construct, make more bits of DNA by PCR, try to express them. There are people who are working on very difficult, poorly understood targets who end up doing this for a year or more.”
There is another way. Dr. Hart’s high-throughput Expression of Soluble Proteins by Random Incremental Truncation (ESPRIT) platform utilizes a directed evolution approach to generate essentially all possible domains found in a single gene, translate them, and screen them for solubility, all in a single, linear experiment.
“We use enzymes to randomly eat away at one or both ends of the piece of DNA,” he said. “The large proportion of this collection is junk—the domain boundaries are wrong, the bit of protein that is produced is nonsense and doesn’t fold up properly so it’s generally insoluble or protealized.” But the hope is that perhaps 1 in 1,000 of these truncated constructs will actually encode the piece of protein that folds up into a stable domain.
The plasmids are all made in a single droplet of just a few microliters, used to transform E. coli where individual bacteria take up single plasmids. Those cells are plated on agar where, during cell growth, an endogenous bacterial enzyme biotinylates a reporter tag on the target protein if soluble, providing a flag for recognition of the rare desired clones. Robotics choose 28,000 colonies per target and place them into the wells of 72 384-well labeled plates.
The contents of the plates are then arrayed onto a membrane, and the plate is frozen down. The membrane is probed with a fluorescent streptavidin to identify positive clones, which can then be taken out of the freezer to be sequenced.
Academic access to the technology is supported by EU funds, with contract research possible for pharmaceutical and vaccine companies.
“If we think that there are well-behaving domains that might be discoverable and that there is no obvious reason why it shouldn’t express in bacteria, we take the project,” Dr. Hart said. “And of those projects—which usually come with a history of failure and frustration, having been worked on in the classical way for many months—more than half actually yield soluble material. The person who has put the energy into doing this experiment comes away with a smile on their face, with something that they want to continue working on.”