Hello fellow scientists. This is more of a request to discuss than a question:
We routinely perform massive-scale (many liters) transfections of HEK293 cell lines, and we routinely notice that these cells begin to secrete HSP70 (definitively identified by MS) in milligram quantities. Secretion levels are anti-correlate with the heterologous protein yield. Did anyone else observe this (I bet you did!) and have you formed any provable theories regarding the nature of the stimuli responsible for HSP secretion? Yes, we have read most (all?) of the papers regarding the paradox of HSP70 secretion (after all it does not have conventional secretion signals). No, the protein is not 'leaking' from damaged cells - our cultures are very healthy for a few days post-transfection.
Would be curious to compare notes.
Does this phenomenon occur frequently regardless the type of plasmid transfected?
I just wonder if any antibiotic resistance marker or the promoter in the plasmid may have some effect on it?
All sorts of plasmids and markers, including marker-less (or to be exact prokaryotic marker only) plasmids. So far we mostly used CMV promoter, to be fair so it can be promoter-dependent.
Secretion of Hsp70 (HspA1A/B) from the healthy cells may occur under a variety of stressfull conditions - mild heat shock being the most obvious one (and the transfection conditions are usually stressfull for the cells). As well as the conditions of heterologious protein expression are also stressfull. May be, the more stressed cells you have, the less is your protein production and the more Hsp70 is secreted from cells. Also, sodium butirate (which is sometimes used to enhance production from CMV promoters) is known to elevate intracellular Hsp70 concentration, which may also affect its secretion.
I think there's definitely a component of cellular stress involved - protein dependent or not it's just hard to say. We use butirate often, but the pesky HSP phenomenon is only with certain proteins/plasmids so that's likely not the cause :) temperature control is a high priority for us so it's not heat shock either. The plot thickens...
Then the question is, are the expressed proteins, promoting Hsp70 secretion, prone to aggregate formation? Do they have any problems with folding, which may cause elevation of Hsp70 expression? To elucidate the question, you may check the presence of the protein of interest in the insoluble fraction of cell extract and check the level of intracellular Hsp70 in cells, expressing various heterologous proteins. Because in the case of non-toxic proteins promoting Hsp70 secretion (in milligram quantities!) you will get an interesting instrument for oncoimmunology.
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