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.”
Shaoqiu Zhuo, Ph.D., presented diaDexus’ work on the human embryo kidney-293 (HEK-293) cell line, which has been widely used to produce recombinant proteins. Many human proteins are cell-type dependent in expression and post-translational processing. “We have developed protein engineering methods for the expression and secretion of recombinant proteins in HEK-293 cells,” said Dr. Wolfert.