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Feature Articles : Apr 1, 2008 ( )
Revolutionizing Protein Expression in R&D Arena
Designing Better Systems to Energize Therapeutic Pipelines!--h2>
The expression of proteins for characterization, therapeutics, and diagnostics continues to be a challenging and complex task, requiring much time and untold expense. The payoffs, though, are well worth it. “Biomanufacturing is a multibillion dollar burgeoning industry,” noted Carlos Miguez, project leader, microbial and enzymatic technology group, National Research Council Biotechnology Research Institute in Canada. New data emerges every day that provides unique insights into methods for producing these proteins.
CHI’s “PepTalk 2008” conference in San Diego earlier this year explored challenges with protein expression, peptide and protein-based therapeutics, and mining the plasma proteome. Various solutions were offered for problems and bottlenecks.
Versabodies as Antibody Copycats
Antibody mimetics derived from nonhuman protein scaffolds offer an alternative to antibodies and small molecules. Volker Schellenberger, Ph.D., cofounder of Amunix, described his company’s microprotein-based Versabody™ platform and the philosophy that has driven it.
The resulting freedom to completely change the product’s amino acid sequence allows Amunix to optimize products for all desired properties such as stability, expression, formulation; whatever the therapeutic area calls for. “The product format is also designed to support optimal patent strategies,” Dr. Schellenberger concluded.
Novel E. coli Expression System
“You look at the number of microbial expression systems out there and wonder why there are so many of them,” Miguez pointed out. “The short answer to that is that, while an expression platform for protein x may work beautifully, when you move to protein y, it doesn’t work quite as well.”
Optimization in Baculoviral Expression System
Jim King, Ph.D., principal scientist for Boehringer-Ingleheim, and his group use the baculoviral expression system for optimization of proteins for crystallography. He calls this expression system the workhorse platform in both industry and academia for the last 15 years. “Until about five years ago,” Dr. King added, “it was really just a production system. In the last five years, it’s really advanced to screening a much larger number of constructs.”
Dr. King’s group has optimized and automated a high-throughput, small-scale baculoviral expression process to quickly and effectively assess protein expression. After one-step affinity purification, the resulting protein is analyzed using a Caliper Life Sciences’ LabChip 90 and is suitable for follow-up characterization to prioritize large-scale expression.
“We have applied this technique to many projects to scan domain boundary variants and point mutants. We actually do parallel small-scale screening, which allows us to use 96 constructs in parallel during one screen, and that screen takes about 10 days to get through all the steps. With the help of Caliper, we’ve created an automated system that does all the steps for us. The system is up and running, working out well, and performing exactly as we had hoped.”
The end game is to make systems easier to use and more accurate, making it easier for scientists to do their jobs, commented Professor Linda King, founder of Oxford Expression Technologies. “People who use high-throughput systems want them to be precise, so they can be used in conjunction with robotic technologies. Also, increasing the quality of the protein is key—as purity goes up, specific activity should also increase.”
Dr. King’s presentation highlighted recent advances in high-throughput, robotic technologies for the production of proteins in insect cells. The new advances include modifications to the baculovirus expression vector, flashBAC, to delete nonessential genes that result in enhanced yield and quality of many proteins. “The development of baculovirus-expression vector systems has accompanied a rapid expansion of our knowledge about the genes, their function, and regulation in insect cells,” said Dr. King.
Baculovirus gene expression occurs in an ordered cascade, regulated by early, late, and very late gene promoters. It has also been realized that the insect host cell has innate defenses against baculoviruses in the form of an apoptotic response to virus invasion. “Baculoviruses counter this by encoding apoptotic-suppressors, which also appear to have a role in determining the host range of the virus,” Dr. King stated. “Also of importance to our understanding of baculovirus expression systems is how the virus can accumulate mutations within genes that affect recombinant protein yield in cell culture.”
Protein Engineering for Diagnostics
“The work we do is not directly related to protein therapeutic applications, but we do research protein-expression technologies for diagnostic purposes,” stated Bob Wolfert, Ph.D., CSO for diaDexus. “What our team has done is optimize methods at high secretion rates. Our first product, the PLAC test, is a simple blood test that measures levels of an inflammatory enzyme that represents a new risk factor for coronary heart disease and stroke.”
“One of the case studies we showed in the presentation is to enhance the expression and secretion of the mature macrophage inhibitory cytokine-1. We have identified one of the rate-limiting steps for the folding of the protein based on a novel glycosylation mechanism and are able to significantly increase the secretion of the mature dimeric form of the protein. This mutation and expression process can be easily adapted into 510K GMP production for our diagnostic products.”
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