“We are trying to reduce the initial cost and time as we figure out the best expression system for a particular protein,” said Dominic Esposito, Ph.D., director of the protein expression laboratory at SAIC-Frederick.
Until recently, Dr. Esposito and colleagues used a more traditional approach, in which after cloning, proteins were expressed with different affinity and solubility tags in various hosts and the best combinations were pursued for subsequent large-scale expression and purification. However, successful scale-up was reported for as few as ~30% from several hundreds of proteins that were investigated.
Dr. Esposito and colleagues recently described an approach known as Purify First, and demonstrated its superiority over the traditional purification method in a study on nine proteins from xenothropic murine leukemia virus-related virus.
Purify First replaces the iterative, large-scale process used previously, which usually went through a series of expensive constructs and hosts before finding the one that works best. This approach is far better in deciding whether and how to proceed with large-scale protein-expression projects.
“Our goal was to develop a technology that explores all possible ways to express a protein at a small scale in a way that is not as expensive and does not take very long, and from there decide which conditions to scaleup. The idea is to minimize the cost and time downstream by exploring these aspects from the beginning,” explained Dr. Esposito.
One of the earliest protein-expression systems was E. coli, but targets are getting increasingly difficult and other expression systems such as baculovirus and mammalian cells are needed, but these have not been in use that long to be optimized and are not as robust. In addition, the amount of protein one can get is not as good as from bacteria.
“It is important to find a system that makes enough protein, and then scale it up, but for many proteins, particularly human ones, this has been a challenge.”
Approximately 50% to 60% of the work in Dr. Esposito’s lab is on baculovirus, which tends to give scales comparable to the ones in bacteria, it handles eukaryotic proteins better, and is cheaper than mammalian systems. “We have spent a significant time optimizing baculovirus expression, and we were able to reduce an expression experiment from three or four weeks to one week or two at most, which is a big advantage.”
“We focus more on the individual proteins and usually work with academia and start-up companies,” said Tsafi Danieli, Ph.D., head of the protein-expression facility at The Hebrew University of Jerusalem.
The protein-expression and purification facilities at the Hebrew University train and help end-users implement new technologies. In a unique mode of operation, Dr. Danieli and colleagues are trying to find solutions for several experimental steps, ranging from cloning and expression to downstream applications, such as activity assays that use the proteins of interest.
Some of the recent projects that Dr. Danieli and collaborators are focusing on include developing baculovirus-based vaccines for veterinary applications, and improving the expression of secreted and nonsecreted proteins in prokaryotic and eukaryotic systems. After an initial stage of working mainly with academia, the core facility started to also work with biotech companies.
“I found that opening our facility to biotech companies infused new applications and methodologies, and allowed us to develop new platforms that benefit and advance academic research.”
To establish a platform that ensures a better exchange of information, Protein Production and Purification Partnership in Europe (P4EU) was established in 2010 as a European network of protein-expression groups that includes protein-expression and purification facilities from the EMBL, Sorbonne University, The University of Oxford, and the Max Planck Institute of Biochemistry.
“Sharing information and reagents is the most important issue in our field, and we are trying to organize a network of several European protein-expression facilities to meet regularly and exchange knowledge and materials that are not proprietary,” Dr. Danieli explained.