Both will probably work. Expression in insect cells is most likely to work well, but will of course be more expensive and time consuming. There's also some slight possibility of getting unwanted glycosylation - which is not necessarily a problem.
I've had good success expressing VHH fragments (single IG domain, as well as some constructs consisting of two IG domains - which are about the same size as your scFv) in *coli *periplasm, using a pelB leader for proper localization. The yield-per-liter is probably not as high as you would see with insect cell expression, but *coli* expression is so fast and cheap that it doesn't matter. (Note that you should not use an expression strain carrying pLysS, or you'll end up with a gooey mess when you try to get the protein out of the periplasm.) For purification of our VHH constructs, we also found that we could recover a good fraction of the protein by simple freeze thaw: resuspend bacterial pellets in PBS or what would otherwise be lysis buffer (50mM phosphate pH 8, 250NaCl, 10% glycerol), freeze overnight, thaw the next day, repellet cells, and purify the protein from the resulting supernatant. This protocol does not liberate all of the expressed protein from the periplasm, but for constructs that express well this may be an acceptable trade off. Freeze-thaw is so quick and easy that it then becomes trivial to process pellets from 12 or more liters of culture at once. - Karl Schmitz On Tue, Feb 17, 2009 at 3:31 PM, Matt Colins <[email protected]>wrote: > Dear all, > > I'd like to express a single chain variable fragment (scFv) for > crystallization purposes, but cannot decide whether I should do it in > insect cells (let the antibody be secreted into culture medium) or > bacteria (let the antibody be secreted into periplasmic space). > > If someone could give me some advice on the two systems for antibody > expression, I would appreciate it greatly. > > Matt > > > > >
