Overcoming toxicity and poor accumulation are common themes in many unsuccessful attempts to overexpress difficult proteins such as multispanning membrane proteins.
According to Genentech’s Hok Seon Kim, Ph.D., senior research associate in antibody engineering, toxicity can be suppressed and cell growth improved to normal by tightly controlling transcription.
“In addition, translation initiation rates play a critical role in proper targeting, insertion, folding, and efficient accumulation of integral membrane proteins in the E. coli membrane,” Dr. Kim said. The protein candidates for expression evaluations are not themselves used in therapy, but serve as potential targets for other therapeutic drugs.
Toxicity to the expressing organism is a problem with many recombinant proteins, especially as the titer builds up. In the case of the transmembrane proteins, Dr. Kim observed toxicity as early as the cloning stage, in the form of restricted or inhibited cell growth.
Many transmembrane proteins appear to be more toxic than, say, antibodies. “It’s thought that their toxicity results from disruption of the biogenesis of endogenous membrane proteins,” Dr. Kim explained.
In E. coli, the availability of native molecular machineries for membrane protein biogenesis is limiting, particularly the protein that recognizes the first transmembrane domain of multitransmembrane proteins.
“This molecule, SRP, must catch that first hydrophobic transmembrane domain as quickly as possible and target it to the membrane with the help of its receptor,” Dr. Kim said. “That particular signal recognition particle (SRP) is limiting in E. coli.”
In other words, during overexpression of transmembrane proteins, E. coli’s endogenous membrane proteins must compete for the limiting SRP to maintain homeostasis. An expression construct with a too strong translation initiation rate would result in a quick buildup of ribosome-nascent chain complexes with missing signal recognition particles and continue to translate in the cytoplasm, resulting in degradation and/or aggregation. Delayed availability of the SRP results in improper targeting and/or insertion, and re-routing to pathways associated with degradation and/or aggregation.
Poor accumulation is the consequence of this constant high level of degradation and/or aggregation in conjunction with inefficient membrane targeting. The traffic jamming of the membrane protein biogenesis pathway results in “sick” cells.
“It is even worse with GPCRs,” Dr. Kim said, “but there may be other factors at work there, that make some proteins more toxic than others among the class of membrane proteins.”
Dr. Kim’s improvement involves a novel promoter with “tight” transcription regulation to minimize toxicity and improve cell growth. Because toxicity and growth inhibition are observed even before induction, expression efforts begin with compromised cells “and the problem gets worse towards the end,” he said.
In addition, Dr. Kim has used a translational leader to control the translation initiation rate. An optimal translation initiation rate would eliminate the traffic jam and allow efficient recycling of the cell’s molecular machineries, resulting in continuous production of heterologous and endogenous membrane proteins.
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.