I am transiently expressing an approximately 30 kDa his-tagged soluble protein in HEK293T cells using a CMV promoter in a pFastBac vector (Invitrogen). This is the first time I am trying to express and purify a protein so what I did was a trial run. I used electroporation to introduce the plasmid into the cells and subcultured them up to 9 days. In this period of time, I collected the supernatant every 2 days and as a result, the total volume of supernatant that I have collected amounts to 250ml. The problem is that I have not successfully adapted my cells to serum-free suspension culture and so I did my trial run in DMEM with 5% FBS.
The cobalt column (Pierce) has a resin bed volume of 0.2ml and a column volume of 1ml. In order to fit my protein sample into the column, I used the 10 MWCO centrifugal filter (Merck Millipore) and concentrated everything into 2ml. However, as a result of FBS in the medium, the residue turned out to be slightly more viscous than the usual medium. I intend to purify the secreted protein in its native state.
The following are my questions:
1) I was worrying about the stability of the secreted protein. Given that the pH of culture medium would fluctuate, would this affect the structure of the protein?
2) Can anyone suggest a better way to concentrate the protein in preparation for purification by the cobalt column?
In the meanwhile, I am trying to optimise the entire set up by doing the following steps:
1) Generation of a stable clone - I inserted a reporter EGFP gene for selection. This EGFP is under the influence of a separate promoter, not fusion protein.
2) I am still trying to adapt the adherent culture to suspension culture using the HEK Plus Serum Free Medium from ATCC. The magnetic stirrer that I am currently using has a lowest speed of 60 rpm. I am trying to get hold of another with 2 rpm.
3) I am optimising the method for plasmid delivery and I am settling on PEI transfection.
All suggestions are welcome.
Hi Johan,
you could concentrate your solution by dialysis with PEG35000 in the outside buffer. In this way you have a better control over the pH when you buffer the outside buffer with e.g. Tris pH 7.5. There are dialysis tubes available in different pore sizes. You could vary the PEG35000 concentration from 10-50% (although 50% is quite viscous). With a small dialysis tube cut off, lets say 10 kDa, the PEG says outside your solution in the tube. Concentrating in this way might take a little longer but you could leave it over night stirring in the cold room without taking to much care.
Hope that helps.
Best Sabrina
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