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Description
Insect cells are a well established system for expression of recombinant proteins. The system relies on the preparation of infectious, recombinant baculoviruses that contain the genetic instructions for protein synthesis. The process of generating baculoviruses involves their amplification to peak infectivity. Plaque formation assays are the traditional method for measuring the multiplicity of infection (MOI) during the phase of viral titer optimization. However, that method suffers from lack of reproducibility and precision due to cell counting and temperature sensitivity. To assess viral titers, we have developed a method based upon Western blot that is more efficient and reproducible and relies on protein expression in response to viral infection, rather than cell toxicity, as a readout. Antibodies are proteins that are generated by B lymphocyte immune cells to bind specifically to foreign antigens. Because of their use in biotechnology and medicine, antibodies are one of the most valuable classes of biological compounds. Here I report a modified and optimized an insect cell expression system to produce milligram amounts of secreted, engineered precursor Fab antibodies suitable for biochemical and structural analysis. The Fab antibodies generated are part of an ongoing study aimed at understanding their metal binding properties and their potential development as engineered metalloenzymes. As part of a separate study, our lab previously reported an x-ray crystal structure of the human IκB kinase β (IKKβ) subunit at 4 Å resolution. Our difficulty in obtaining higher resolution diffraction data is likely due to inherent flexibility of the IKKβ dimer leading to difficulty in stabilizing one conformational state. In order to solve this problem, we have designed a monomeric form of IKKβ that is engineered to minimize its flexibility. As part of an effort to produce engineered human IKKβ subunit proteins for high resolution x-ray crystallography studies, I have used the baculovirus expression system to produce highly pure monomeric IKKβ in milligram amounts.
