I was expressing a protein in Ecoli BL21 and did the following purification steps:
1- GST purification (Pierce™ Glutathione Agarose 16100)
2- GST tag cleavage (Presscission protease: Genscript Z02799)
3-endotoxin removal ( Pierce 88274 )
4-FPLC purification
5-buffer exchange to PBS by 30kd filter (Millipore UFC803008 )
The final protein purity is above 95% dertemined by SDS-PAGE. I still find it is toxic to cell even after incubating 30 min. A commercial protein as a positive control doesn't have this tissue.
Any one has some comments that i missed something to make the protein ready for cell culture?
Hi,
try using ion exchangers for additional purity, and try to exchange buffer using gel-filtration in order to achieve 100% PBS in your protein prep. You can even dialyze your protein against PBS before gel filtration to be absolutely sure that no toxic chemicals is left in your final prep.
Best of luck!
Could you do the following to determine the source of the toxic contaminant:
do the same thing for pure BL21 culture (without your protein) and pure DI water.
then you would know whether the toxic contaminant is from BL21 or the reagent of purification.
Dear Yong
just 2 questions:
1) Did you measure the endotoxin at the end of the process to see if your endotoxin removal works well?
2) Which was the buffer prior the final buffer exchange and how many washes (from which volume to which volume) did you performed with the 30KDa filter to achieve complete buffer exchange?
best regards
Manuele
Thanks you guys for the suggestion. For the endotoxin, I have tried co-treatment of LPS with the commercial protein from 100ug/ml to 30ng/ml. I didn't find any significant influence on the cell viability for a 30min incubation. This result should exclude the toxicity form endotoxin. I used a tris.hcl buffer(50mM, 150mM Nacl, pH 7.0) as the base of FPLC and then change to PBS by 30kd fliter. A extensive dialyis by PBS and ion exchange might help, but I am confused what component in the protein causes the toxicity to cells only for a 30min incubation.
Tris is quite toxic, but it is not excluded that other contaminants (proteins, nucleoproteins, hydrophobic stuff, aggregated proteins, etc.) kill the cells. I assume that FPLC step is gel filtration, so the easiest would be to simply load your sample after step 3 on column equilibrated with PBS. And then you will know if its Tris or something else.
and yes, check if pI of your protein would allow it to be soluble in PBS (pH 7.4).
Best of luck!
For your SDS-PAGE analysis did you run the protein in both reducing and non-reducing conditions? Your protein might be aggregated which could cause the toxicity.
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