My protein have an Tm of 65℃ after elution with 200mM imidazole ，concentration is 0.2mg/ml,Then I concentrated protein by Millipore 30K ultrafiltration tube to an concentration to 2mg/ml, then I aspirate some for thermal shift assay ,then I didn't get an S shape signal curve and signal is decrease with temperature which means my protein loss it 3D structure , interesting ,the protein have an PH about 12,but the filtered buffer is still have Ph 8.0,As I detect by PH test stripes, My buffer is 50mM NaPoi PH 8.0，300mM NaCl，0.1mM TCEP ,5% glycerol. Did anyone met the same problem or have some suggestions to explain this, thanks all
Hi Liao
I would suggest following :
1.Reducing salt concentration to 150mM NaCl or lower
2. Spining at lower rpm and at 4-10 degree celsius
Use of 5% glycerol may be making your protein solution viscous resulting in longer time to concentrate thus increasing shear exposure to the protein resulting in unfolding. Also see if your buffer pH is very close to the IEP of protein.
Hi Anshul, Thankyou for your advise, I change buffer to 25mM HEPE pH 7.5 ,150mM NaCL with 1mM DTT and spin at 2000rpm and resuspend 10min each time, but after 3 times PH still become higher then protein become unfold in thermal shift assay,
If pH becomes very high and that could be causing protein unfolding... It will be good to use high concentration of HEPES buffer to provide good . Not sure why DTT is being added. DTT usually breaks disulfide bond which can lead to loss of protein structure. May be DTT is causing change to protein structure.
Hi Anshul, Thank you, it may not because the thiol or TCEP reductant make it loss structure ,The lysis buffer(50mM NaPoi PH 8.0 150mM NaCl and 0.5mM TCEP or 2mM β-Me or 0.5mM DTT) I use to lysis bacteria and binding to His Hitrap HP column (GE) then elution with 200mM imidazole still have a right fold with Tm=65℃, So I think DTT is OK
Hi Liao, then it is most probably that your protein is extremely shear sensitive. I will say try Crystallisation to concentrate your sample if you can.
Hi Anshul ,My protein is PHGDH full length with 6His tag and TEV site in N-terminal construct in pet30b vector ,the pI=6.41 of protein calculated by ExPASy ProtParam, condonplus(DE3 ) which add rare tRNA useful to expression mammalian protein was used ,When OD600=0.4, lower temperature to 18℃ and further incubate for 1h，Finnally add 0.4mM IPTG ,incubate 12-14h then harvested, all most papers use to expression PHGDH is pNIC28-Bsa4 vector but I didn't have at that time, fortunately Now I get it from addgene, And I want to try the same condition as papers instruct.
Back to the protein,
I tried to use 1ml HiTrap Q HP 1ml column to concentrated ,But what make me crazy is the protein precipitate in the top cap of the column even at low concentration about 0.1mg/ml total 20ml(the manual said the column can bind 10mg protein ) ，protein is stable in 50mL Tris PH8.0 50mM NaCl(because after I use the buffer to desalting imidazole ,The protein still have an Tm=55℃，which is lower than elution for Ni containing 200mM imidazole ,And I don't know why ) Then I used to load Q column.
Anshul, I am so interesting in your idea of Crystallization to concentrate, Can you offer me some reference or protocols about this I didn't known this method at all ,And I so looking forward to try, Thank you so much.
Hi Liao I do not specialise in Crystallisation so can't refer you to any protocol. It would be better if you speak to a lab which works on protein structures/biophysics. They will be able to help you better as you need a specific freeze drying equipment for this.
- Badges
- Science method