I'm trying to express secreted recombinant proteins in P. pastoris. I'm using the Pichia pink strains from Invitrogen. I've identified a few clones that express my proteins intracellularly (i.e. I can see the proteins in western blots prepared from lysates of the cell pellets), but I'm still trying to identify clones that secrete my proteins into the medium.
I've grown a number of clones containing expression constructs with different secretion signal sequences in small scale cultures in BMMY medium and collected the supernatants. I've tried to analyse these supernatants for the presence of my proteins by SDS-PAGE and western blot, but the total protein concentration in the supernatant seems to be so low that I don't see anything on the gels. I've tried to concentrate the total protein from the supernatants by precipitation with ethanol, which yields a large, brownish pellet, but this pellet refuses to redissolve, even with heating to 95 C in urea buffer.
Try precipitating the supernatant culture with Ammonium sulphate
Hi Scott
Make sure that your protein inside the cells or extracellular. If it is inside cells, you need to do cell breaking up after that you can try fractionating the protein by Polyethalyene glycol or may be with Ammonium sulfate at different concentrations
From your explanation, it seems the expressed proteins are intracellular proteins. if yes, you need to first break up (lyse) the cells..there are different methods for that (sonication, high speed fractionation, hypotonic solution etc). But if the protein is expressed extracellularly, you can precipitate using solid ammonium sulphate gradient.
Good luck!
I should probably clarify my question. I am trying to express secreted proteins. I have clones carrying expression constructs with various secretion signal sequences. However, so far I've only been able to find my proteins in the cell pellets. What I want is to do is find them in the culture supernatants. On the basis of some dot blot immunoassay results I suspect that the proteins are being secreted a very low levels, but the concentration seems to be so low that I can't see the band in western blots. This is why I'm looking for a way to concentrate the protein in the supernatant. I'll try ammonium sulphate.
Hi Scott Godwin,
Have you considered concentrating the secreted protein using ultra-filtration? There are nice concentrators/ultra centrifugal filters which you could try depending on the molecular weight of your protein of interest.
Good luck!
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