September 15, 2011 (Vol. 31, No. 16)
Richard A. A. Stein M.D., Ph.D.
Technology Evolves and Focus Shifts from Process to Dissecting Pathways of Interest
Expressing recombinant proteins is one of the most challenging, but at the same time most important tasks in the life sciences. Irrespective of the specific protein and the downstream applications, some of the most significant questions involve identifying the best expression system, finding the ideal tag, and characterizing the optimal set of conditions to generate a functional protein. This process is often lengthy and costly, particularly when multiple combinations of expression systems and conditions need to be explored.
A protein expression platform created by Pfenex that is based on the Pseudomonas fluorescens strain MB101 was designed to address some of these challenges. “It allows the biology to tell us what expression strain to use when producing a particular protein,” said Bertrand C. Liang, M.D., Ph.D., CEO.
The Pfenex Expression Technology™ platform combines unique host strains and expression strategies, such as specific chaperones and protease deletions, to generate thousands of expression strains that can be analyzed in parallel at the preclinical stage of development. “It is particularly useful for difficult-to-express aglycosylated proteins,” Dr. Liang added.
Initial protein characterization starts with small-scale expression that is followed by larger scale cultures grown under various conditions. The possibility to rapidly test specific strains for their ability to express a protein of interest makes it an attractive tool to generate new therapeutics particularly when there are time constraints, for example, when a new flu vaccine is required or for biodefense applications.
Pfenex showed its ability to deliver on such projects when it was chosen to participate in the DARPA/DTRA-sponsored Accelerated Manufacturing of Pharmaceuticals, or AMP, program to develop technologies to deliver pandemically relevant clinical doses of a vaccine in a previously unrealized goal of less than 12 weeks.
The AMP program culminated in a “live fire” test in which the agencies challenged Pfenex to express and produce the influenza H1N1 hemagglutinin protein, the active antigen in the pandemic flu vaccine. Pfenex scientists, through their parallel strain construction and processing technologies, successfully produced high-titer, high protein quality expression strains and along with collaborators purified the first clinical lots well within the 12-week challenge period.
“Making proteins is hard. A platform that is high throughput and robotically enabled is the key, as it allows many different conditions to be explored at one time, to determine expeditiously the best approach to express high titers of quality protein in an active, soluble manner,” emphasized Dr. Liang.
“The technical field has made tremendous progress in overcoming what we once thought were insurmountable challenges,” said Blaine Pfeifer, Ph.D., assistant professor in chemical and biological engineering at Tufts University. At CHI’s recent “Protein Engineering Summit” Dr. Pfeifer talked about his lab’s efforts to implement an optimized expression system and reconstitute polyketide and isoprenoid natural pathways in E. coli as an expression host.
While significant efforts in the early days focused on producing a protein of interest in E. coli expression systems, the field is now taking a step further. “At present, we need to produce several proteins in their active form that can, at the same time, work together to engineer the small molecule formation in bacteria.”
Recently, Dr. Pfeifer and colleagues developed an E.coli expression system to synthesize erythromycin A, an antibiotic that is produced by the actinomycete bacterium Saccharopolyspora erythraea and requires over 20 enzymes.
In collaboration with Gregory Strephanopoulos from MIT, Dr. Pfeifer uses a similar approach to produce early-stage intermediates of taxol, a promising anticancer drug. Taxol was originally isolated from the Pacific Yew (Taxus brevifolia), but isolating it from its natural source is both economically challenging and ecologically destructive.
The plasmid copy number, the co-expression of chaperones, and the choice of promoters and specific strains are some of the major considerations when expressing foreign genes in E. coli. All these technical aspects are important for the coordinate expression of over 20 genes that are needed for an active natural product pathway.
“This resonates a lot with general challenges that we face during protein expression,” said Dr. Pfeifer. An additional consideration is that the total number of genes in these pathways exceeds 20, and some are larger than 10 kb, exceeding by far the average E. coli gene size.
Therefore, one of the biggest challenges is whether the new host can accommodate these genes, particularly when they are expressed together. “We often don’t pay enough attention to the optimal level of gene expression and to the metabolic burden on the cell. These are some of the next steps that we need to address, as we tune the level of protein expression and metabolites to maximize the enzyme activity and the flow of carbon through our products of interest.”
“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.
“We commercialized a microfluidic chip analysis application that performs capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) to rapidly characterize proteins,” noted Mark Roskey, Ph.D., svp at Caliper Life Sciences.
The platform, LabChip GXII, performs high-throughput capillary electrophoresis in 96- or 384-well plates. In addition to processing 96 samples in an hour, LabChip GXII provides information on protein size, concentration, quality, and purity, by using only miniscule amounts of material, as little as 2 µL, according to Dr. Roskey.
“This is a very rapid way to characterize a protein that is being expressed or developed as a therapeutic, and one can do it in very high throughput.” The platform can provide information regarding protein dimerization, aggregation, and fragmentation, can be performed under nonreducing or reducing conditions, and is >70 times faster than more traditional separation technologies.
One particularly important application is the characterization of glycans. Obtaining an assessment of the glycan profile of proteins such as antibodies is crucial because this modification affects their functions.
“We developed a very rapid kit that cleaves off the sugars and uses the same technology to analyze which sugars are present and which ones are absent from a protein of interest,” explained Dr. Roskey.
Glycan profiling is important, as carbohydrate expression patterns often have biological relevance. For example, certain glycans were implicated in tumor progression and in the adhesive interactions that occur as part of tumor metastatic dissemination, and depending on the glycan and cell types, they may also have tumor suppressor effects.
Protein expression has witnessed many recent changes. Technical advances have made it possible to obtain proteins and protein complexes that years ago were not available for scrutiny, and high-throughput platforms have allowed multiple expression systems to be tested in parallel, and significantly faster than before. These developments are reshaping the life sciences, and promise to shift the efforts from protein expression toward dissecting the biological processes and pathways of interest.