Hi All,
I am trying to purify a recombinant His-tag protein from insect cell (baculovirus expression system) and my protein is a nucleus protein. Previously during clone selection stage, I used buffer containing 1% Triton X-100 and 0.1% SDS to lyse the insect cells and can see strong protein expressing band. For purification by Ni-NTA resin (His-tag purification), I used I-PER Insect Cell Protein Extraction Reagent for breaking up the cells but it doesn't seem to release as much of my target protein as I would like.
Can anyone who has successfully purify recombinant protein from insect cell give me suggestions as to the best way for breaking the cells for releasing the protein into solution. For instance, is it possible to just use sonication to break open the insect cells?
Thanks,
Lyra
Sonication can be used, but typically isn't necessary. A dounce homogenizer may also be used. It is also possible that a large fraction of the protein is insoluble, as that is common for overexpressed proteins.
A homogenizer can be used and is best for large volumes. I typically use sonication for my preps. Pulse 10 sec on/40 secs off for a total pulsing time of 2-2.5 mins and keep your sample in plenty of ice. That should break your cells perfectly. Remember to add protease inhibitors before breaking your cells. You may need to just your power level, I typically have an output of around 65 watt. Best of luck.
hi Lyra
i did his-tag purification with BL21 cells and usually i got 3-4 mg/ml of protein concentration with triton-x and sonication. although it is difficult to get higher concentration in insect cell, homogenization than short burst treatment of sonication may help you
Thank you all for useful tips about breaking open insect cells. Sorry I should reply earlier but I was too busy last week with other experiments!
Tomorrow I am going to try breaking insect cell in Na phosphate buffer (pH=8) containing 500 mM NaCl and 10% glycerol using sonication at 80% amplitude 10 sec on 1minute off cycle for 10 cycles.
I would like to try dounce homogenizer but unfortunately we don't have one. So I will use sonication instead.
Best,
Lyra
Hi Wei,
At the end, I used just sonication and it works.
But I found that insect cells have much lower expression than E. coli and sometimes the protein partners can be co-purified or form complex that prevent His tag from binding to the Ni resin. (ex. our human HDAC1 can form complex with other HDAC2, HDAC3 in insect cells and not binding to Ni resin)
I am not a big fan for insect cell expression but sometime you just have to use it. Now I try expressing in E. coli first if possible since insect cell really doesn't boost expression much.
Best,
Lyra
Thanks Lyra, WE are going to look at modifications of the protein, so insect cell seems a best choice by comparing to other mammalian cells. we have already done expression in E.coli and we didn't have problem for purification. maybe that is the case that the protein form the complex.
Thanks
Wei
Sonication will help, but I would try it with the urea-thiourea combination for the buffer. These compounds can be removed later by dialysis.
- Badges
- Science method