Hi Sutapa, Slow expression did work in my case. I had to induce with 100uM IPTG, and grow cells at 10C for 48 hours. It left at least 50% of expressed protein in soluble fraction.
Best, Swarna On Wed, Apr 5, 2017 at 1:52 AM, AJV <[email protected]> wrote: > 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. >
