Hi Dima, >Can anyone comment on availability of Archaea expression systems? >Based on available structures, it would appear that their chaperons >are much more similar to eukaryotic one than anything present in >eubacteria. For a long time, I've been puzzling over why there is >still no popular/workable Archaea expression system. Reason?
This one caught my eye recently: https://drum.umd.edu/dspace/handle/1903/3285 Methanosarcina acetivorans There is one more system I am aware of - the one based on the pDT2 plasmid for Haloarcula marismortui and related species. Don't have a reference on hand - too lazy to look up. The likely reasons as to why archeal expression systems are lagging behind pretty much every other type is a) many (most?) archea require crazy media for high-density growth b) transformation of archea is difficult (most of them need to be spheroplasted), c) there aren't many (nearly any) stable high-copy plasmids, and d) the knowledge of archeal promoters is way less extensive than that of bacterial, yeast, etc. They're coming, to be sure. Another 5 years or so, I'd guess. >I've compared Sf9/Sf21 and High5 cells for many, many baculoviruses >and have yet to find a difference that would ultimately matter. We've had the opposite experience. In a considerable number of cases Sf9 and Sf21 cells were much more successful than the Hi5. In a very few (but significant!) number of cases Hi5 were much, much better. We suspect for at least a handful of cases that the Hi5 cells expressed *too much* of the protein of interest, resulting in almost no soluble protein (but tons of insoluble). >All of them are essentially equivalent = pathetic, as far as >overexpression is concerned. Mammalian cell cultures have reported cases of secreted expression well into hundreds of mg per liter, for stably transformed cells grown in media-perfused porous substrate. Granted, you have to a) know what you're doing b) have enough money to keep trying again and again and c) hunt around for the well-expressing clonal lines but I personally wouldn't call this 'pathetic'. >Dictyostelium discoideum (slime mold) deserves honorary mention >here. Yeah, I was going to try it some day but never had any free time left :) Sounds interesting. Not too many (any?) strong promoters though. >IMHO, if you don't get *any* soluble expression at all in minimal >medium expressing at 16C ... I would slightly expand Dima's statement: if you test at least the following in E. coli, and get nothing then you're getting into a game of diminishing returns: Rich medium - TBIIx1.5 + 0.8% glucose + 1.6% glycerol at 20C Poor medium - M9 or similar + 0.8% glycerol at 16C (like Dima said) Try these two media with a simple His-tag and with a His-MBP- fusion protein. So that's a total of 4 experiments. If in both cases you don't get at least a trace of soluble expression then you may want to try other systems. Note that if you have signs of toxicity - that's actually encouraging because typically in order to be toxic the protein has to be at least partially folded. Cheers, Artem
