On 3 Jan., 06:44, Tiwari <kstiw... from gmail.com> wrote:
> Hi Guys,
>> I am expressing the recombinant protein 6xHis tagged at C' terminal
> secreting in YP-Galactose media by K lactis. My problem is whenever i
> try to purify by using Ni-NTA column, it turns the column white as if
> supernatant is chelating Ni from the column. I tried to condition
> supernatant by adjusting pH 8.0 and Salt upto 1M with no success.
> Please suggest me what should i do. Ammonium sulphate precipitation
> result in inactivation of protein. i cant do dialysis economically to
> huge culture volume. so these will not be a good option for
> conditioning media.
>> Thanks!
Hi Tiwari,
I have had the same problem with Baculovirus System. Media contains
both a chelating agent and a imidazole-like molecule (probable
histidine in high amounts) :-(. For a long time we have dialysed
samples for three days against 5 x 8 L buffer. Such a waste of water,
time and chemicals. But there are smart solutions.
You could try ion-exchange column to concentrate protein and get rid
of oppositely charged contaminants. Then the sample volume would be
lowered to 10 - 20 mL, which can be dialysed or desalted otherwise. If
you purify an enzyme and have an assay for detection, this can quickly
be optimized.
The best solution is a CrossFlow machine. We just bought one in my
lab. It takes in fx. 1-600 mL sample (we only have 600 mL as max, but
machine can take more), concentrates to 32 mL and diafiltrates (10 kDa
cut-off) with 160 mL buffer. This results in approx. 45 mL
concentrated sample with a 95 % recovery of protein and a 95-98 %
reduction in small contaminants, just ready to go to the Ni-NTA
column. All done in 3 - 4 hrs. Nothing can beat that.
- Regards
Gormicon
