Good morning everybody
I'm being very headache with my protein purification. Please give me any option you can.
1/ I did construct 6 proteins, including:
-Flagellin A (FlaA), Flagellin B (FlaB)
-FlaA-PspA, FlaB-PspA (fusion proteins)
-FlaA-TTFC, FlaB-TTFC (fusion proteins, TTFC: fragment Hc of Tetanus toxoid)
All proteins were expressed well in E.coli ER2566/pTYB12 (Intein tag) and purified. I did get purified FlaA, FlaB, FlaA-PspA, and FlaB-PspA with accepted purity. My problem is with FlaA-TTFC, FlaB-TTFC. There are very strong bands contaminated with my final products (please see the attached picture)
2/ I have applied the following purification protocol
- 1 litter culture (seeded with 10ml of overnight culture)
- Shaking incubation at 37oC, 4hours (OD600nm= 0.9-1.0)
- Induction: IPTG with final concentration of 0.4mM, 16oC, 16hours
- Collect the pellet, suspend in 50ml lysis buffer (Tris-HCl 7.5, NaCl 500mM, EDTA 1mM, Triton 0.1%, PMSF 0.02%)
- Sonication on ice (7minutes, 5'' on, 5'' off)
- Centrifugation (18000rpm, 30min-1hours), collect supernatant
- Loading on Chitin bead column with speed of 6 seconds/drop
- Washing step: at least 1 litter washing buffer. Washing buffer: Tris-HCl 7.5, NaCl 500mM, EDTA 1mM, Triton 0.1%
- Cleavage with buffer of Tris-HCl 7.5, NaCl 500mM, EDTA 1mM, DTT 50mM, at room temperature, 40hours.
3/ I did try some modifications with hope to get purer protein, but all failed, such as:
- Try different IPTG conc: 0.1mM, 0.25mM, 0.4 mM
- Temperature for induction: 12oC, 15oC, 18oC
- Washing step: increase Triton concentration (0.2%, 0.3%, 0.4%) NaCl concentration (750mM, 1M). I even used 2 litters washing buffer for one column.
- Cleavage: + DTT concentration (5mM, 10mM, 30mM, 50mM)
+ Temperature: room temperature, 4oC
+ Time: 16hours, 24hours, 40hours
4/ I also designed one linker (15 amino acid) between flagellin and TTFC with hope to make the fusion more stable and well folding. However, the purity still did not get any improvement.
Actually I think TTFC is causative agent of this problem because my other proteins do not meet this problem. However I could find some papers that showed very pure protein of TTFC as well as its fusions to other proteins.
Until now, I can not find down any more solution for this. Please help me with your kind heart.
Thanks so much