Hi
Now I'm trying to express and purify human protein using E. Coli. The protein size is 14 kDa and pI is 8.2. I made two expression vectors carrying the target protein using pGex6p1 for GST-fusion and pCold for His-tag fusion. Though, both tagged protein were mainly expressed in insoluble fraction, I got small amount of purified protein from soluble fraction after affinity purification. But the amount of protein from the culture is not enough for structural analysis.
Below are the conditions I tried for both vector.
Strains:BL21(DE3), Rosetta (DE3)pLysS, Shuffle
There was no significant difference between BL21 and Rosetta about expression. Shuffle showed much more growth than other strains but a lot of cells died after O/N culture and amount of the protein from the cells was small.
Conditions:On all vectors and strains, 16 degree at OD600=0.4 induction was better than 25 degree. IPTG concentration: 0.1 mM and 0.01 mM were better than 1 mM. Should I lower the temperature less than 16 degree, such as 10 degree?
Medium:LB medium.
Purification:Small amount of GST-fused protein was obtained from soluble fraction with good purity and GST could be cleaved by protease. The amount of His-tag fusion protein from pCold vector was not reproducive. Sometimes it contains a lot of non-specific proteins even after similar careful washing.
Now I'm making pEt22b based C-terminus his-tagged vector.
And I also bought chaperon set from TAKARA and I'll try them.
How can I refine?
Any suggestion, please.
Hello, if the main problem is that protein is in the insoluble fraction I think that the best solution is to use a tag in order to try solve this problem. In my hands a MBP tag works quite well for the soluble expression of proteins, better than GST. If to cleave the protein doesn't give problems it's better to use a tag like MBP or similar than a C-terminus his-tag. Chaperon set could work too, but perhaps just induce heat shock proteins with ethanol and 4°C before induction can also help you.
Hi,
Try auto-induction expression. In my case, high solubility and high yield with any tag. IPTG induction also gave insoluble constructs and low yield in my case. AI also gives you more options if structure analysis is your goal.
You may try minimal medium at different temperatures and different expresion time (if you want to stick with IPTG
If non of this works, use SUMO tag, or MBP as Jaime said.
Let me know what happens !
Hi,
Try insect cell or mammal cell. Maybe your target protein should have some modification.
Hope this helps!
In addition you could try the new cell line from SGI-DNA (https://www.sgidna.com/vmax-express.html). It is a vibrio bacterium that they claim can increase solubility of proteins. The good thing here is if you can grow E. coli you should be able to easily use this cell line as most vectors used for E. coli are translatable to this cell line. They do say you need their special media to grow which is expensive. However, they say LB miller will work but not a efficiently.
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