Hi Sutapa, The input from other commenters has been great, so I'll stick to discussing only the portion of your question regarding baculovirus.
In our experience, codon-optimization has resulted in little to no improvement of total protein yields or protein solubility in baculovirus. We routinely work on challenging targets (mammalian membrane proteins), and while we don't have mounds of data on the subject, we have expressed enough eukaryotic protein families to say this with some confidence. Our workaround to bad expressers has been to screen orthologs of the protein of interest, using life's natural codon diversity to our advantage. That being said, my suggestion would be to attempt baculovirus again, as there must have been a good reason for you to try it in the first place. Not knowing whether your gene is eukaryotic, specifically mammalian, or if post-translational modifications could be important; but if any of these are the case, baculovirus would be one of the best systems to pound on. We test several variables that always result in "seeing" some amount of protein expression with baculovirus (depending on detection method) -- time, temperature, and MOI. Once high titer / low passage virus is obtained and titered (can't stress the importance of titering enough), I usually set up six 50mL suspension cultures of Sf9 cells and infect them at 2x10^6 cells/mL, using MOIs of 0.2, 2.0, and 20.0, in duplicate. After 24hrs at 27°C, I place three of the cultures at each MOI in an identical shaking incubator at 20-23°C, and leave the other three at 27°C. I then remove ~5mL of culture at various time points (about every 12 or 24hrs depending on scheduling), up to ~120hrs for all six samples. Then, crack the cells and use your detection method of choice (GFP, Western, Coomassie) to assess expression. This workflow can be repeated using other insect cell types (Sf21, Tn5), as they all will vary in their protein expression capabilities. Hope this answers part of your question, and results in some quality sample. Best, Alex _____________________________________ Alex J. Vecchio, Ph.D. Postdoctoral Fellow University of California, San Francisco Department of Biochemistry & Biophysics Laboratory of Robert M. Stroud, Ph.D.
