Endogenous 3C-like protease activity in HEK cells

Hi All,

Has anyone scereted proteins from HEK cells with a 3C cleavable tag? Our problem is that we have good evidence for expression and we have been able to purify a particular c-terminally OneStrep tagged secreted protein but when we add a 3C site for removal of the OneStep tag we are unable to bind anything to the Streptactin columns. So either the 3C site prevents secretion (that we think is unlikely) or the 3C site is cleaved by an endogenous HEK protease either before or after secretion.

Any ideas anyone?

Thanks

Nick

Posted on 04-Oct-2012 13:52 CEST

Hi Nick,

we use pHLsecFcHis plasmid from Radu Aricescu from time to time, it contains 3C site between the protein construct and Fc IgG fragment and we purify the expressed fusion proteins normally via His-tag and gel filtration and later cleave them by 3C protease prepared in-house and everything seems prety normal, e.g. the overall yields are similar to other Fc IgG-tagged constructs with TEV cleavage site, and this is true for 293T or 293E or 293S GnTI- cell lines and different culture media. No idea to your observation, sorry...

Posted on 30-Oct-2012 17:44 CET

Hi Nick,

We have the same experience as Ondrej mentioned with a secreted protein containing a C-terminal Fc fragment. The protein could be cleaved by 3C-protease when the protein was bound to ProteinA sepharose. I don't recall that there were particular issues with the expression of the protein. The protein was expressed in HEK293 Flp In cells.

Also no clue what could be happening in your case..

Best,

Patrick

 

Posted on 31-Oct-2012 11:31 CET
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