Some proteins are easier to express than others. For some, just clone them into an E. coli vector and let the bacteria do the work. But for others—such as toxic proteins, membrane proteins, glycosylated proteins, and hydrophobic proteins—getting them to express, fold, and function or crystalize can present a challenge.
Scientists at CHI’s “PEGS” conference, held last month in Boston, were keen to talk about some of the difficulties they faced in expressing challenging proteins. Fortunately, they were just as keen to talk about the solutions they came up with.
Typically, when researchers want to express a protein they turn first to E. coli: transfect the bacteria with the appropriate vector, grow it up, plate it out, select colonies, and grow up cultures of the selected clones. Then the fun of purification begins.
Yet for work on an analytical scale, protein can be generated without the need for cell culture (though this, alas, doesn’t alleviate the subsequent need for biochemistry). Several in vitro translation systems based on bacterial, yeast, plant, insect, and mammalian cell extracts exist to turn DNA or RNA into protein.
These all have the added advantage of being able to express proteins that may be toxic to a cell. Each, of course, has its own pros and cons as well—bacterial- and wheat germ-based systems have higher yields, but these and the rabbit reticulocyte systems aren’t capable of glycosylating the resultant proteins, for example, while insect cell lysates introduce insect-specific post-translational modifications.
There are advantages to expressing human proteins using a human system, foremost among them are that the products will be properly folded and properly post-translationally modified, explained Brian Webb, platform manager at Thermo Fisher Scientific. Many academics have published on making a cell-free protein-expression system from human cell lysates, but to date there is only a single commercial product line based on research from the Riken Institute in Japan and licensed by Thermo.
Thermo’s kits make use of a T7 promoter and an internal ribosome entry site, so that it is not necessary for the resultant RNA to be capped before being translated. One set of kits, based on HeLa cell extract, has been optimized for high yield—currently promising up to about 40 ug/mL in about 90 minutes. Data was presented at “PEGS” indicating that yields of several hundred µg of recombinant protein per mL of reaction are possible, and Webb said that such higher-producing kits should be available in the fall. A second set, based on a hybridoma, is optimized for the expression of glycoproteins.
Multiple proteins can be expressed, potentially allowing protein complexes that have more than one subunit in the same reaction, which could “allow those subunits to form and carry out their function,” Webb added.
Later this year Thermo will introduce a series of expression vectors that include C- or N-terminal flag, HA, or myc tags, to complement the currently available HIS tag vector. Fusion vectors encoding GFP are also in the making.
Dare to Compare
For production quantities, cell culture/fermentation is still the way to go. And, although biopharmaceuticals have been on the market for more than 20 years, those products have been produced by only a handful of expression systems, said Georg Klima, Ph.D., head of microbial process science at Boehringer Ingelheim Biopharmaceuticals.
Not every system can handle every protein. Dr. Klima was working on a dimeric Fab antibody fragment that he suspected would be difficult to express—the target it binds to was quite hydrophobic, and looking at the Fab’s variable regions indicated it might tend to aggregate. He decided to do a side-by-side comparison of different systems.
In one arm of the test, light and heavy chains were expressed separately with good titers as inclusion bodies and could be refolded separately, but could not be assembled together, nor could they be refolded together. He found no expression when attempting to express the Fab in the E. coli periplasm. And similarly, no expression was seen when trying to get the methylotrophic yeast Pishia pastoris to secrete the Fab into the supernatant.
Only the Pseudomonas fluorescent expression platform, developed by Pfenex, yielded a properly folded, biologically active dimer. “That confirmed that we had a difficult-to-express molecule,” he noted.
Pfenex can create over 1,000 individual strains. This particular project combined 20 different plasmids with certain genetic elements on them with 50 different host strains also with different properties such as protease deletion. These were screened on the 96-well plate scale, and those strains yielding the highest active Fab:target binding titer by bio-layer interferometry (BLI) analysis were chosen for fermentation scouting.
For this part of the study, 24-unit, 4 mL bioreactors allowed a broad range of induction conditions to be compared. The best strains and conditions from this stage were then moved into a conventional 1 L reactor—“which lets you estimate what will happen in a production bioreactor,” he noted. The process from strain construction and screening to fermentation confirmation, scaleup, and purification took about 10 weeks.
The resulting material had high levels of purity, excellent solubility, and high affinity. “Overall it was a successful study for us,” Dr. Klima remarked. While a traditional E. coli fermentation is still his first choice for protein expression, Dr. Klima thinks that the Pfenex system will be very useful with other hard-to-express proteins.