At Manufacturing Scale
Expressing difficult proteins is most challenging in a production setting, where the manufacturability of stable, correctly folded proteins often determines success or failure.
Speaking at the “PEGS” event this April, Ian Hunt, Ph.D., head of protein sciences at the Novartis Institute for BioMedical Research, hashed out the pros and cons of E. coli and baculovirus expression as it relates to difficult-to-produce proteins.
Baculovirus expression requires insertion of DNA into a viral genome, transfection, and two or three rounds of amplification to produce enough virus at the correct titer.
“With insect cells, it typically takes two to three weeks before you can determine if your protein is expressing,” Dr. Hunt said.
E. coli is a more efficient and accessible system than insect cells, in part because it does not involve a viral component. Cells are transformed directly and begin expressing protein, of which measurable quantities become available within a few days, sometimes overnight.
“E. coli is much faster, and much more amenable to high-throughput expression testing,” Dr. Hunt said. It is possible to fragment very difficult protein targets into 10 or 20 smaller domains, express them in parallel in E. coli, and see which is the most stable and prolific. “It doesn’t take much time, and does not require a lot of hands-on time. But the same process is quite laborious in insect cells,” he added.
Dr. Hunt described the relative benefits and capabilities of the two expression systems as a tradeoff.
“E. coli is fast and easy, but it’s most applicable for relatively small, easy to produce proteins, whereas insect cells can produce larger, difficult proteins but it takes a lot longer.”
Insect cells are capable of expressing large, difficult, multidomain, intact proteins that E. coli cannot. Once a protein reaches 60–70 kD in size, its soluble expression becomes problematic in E. coli.
Increasingly, pharmaceutical and biotech companies are turning to difficult protein targets that, more often than not, are difficult-to-express membrane proteins. Breaking those proteins up works to produce small, easy-to-manufacture subdomains as targets for drug discovery.
Another topical target of pharmaceutical development is disruption of protein complexes. One way to obtain complexes is to express and purify the relevant proteins individually. Dr. Hunt described an approach, co-expression, by which two or more viruses transfect insect cells simultaneously, resulting in the production of an equivalent number of proteins within the same insect cell culture.
After simultaneous purification, the proteins are in the proper proximity for complexation. Disruption might occur through direct inhibition of binding sites or by allosteric interactions. Or, one could label one protein with an affinity tag, purify the complex as one “molecule,” and use it in drug screens.
“Co-expression is one of the major strengths of insect cells,” Dr. Hunt observed.