Can anybody send me detail protocol for expression and purification of protein from MBP vector?
Hi
If your choice of vectors are pMALc5X/pMALc2X you will get a clean protein with two-step purification(Affinity and SEC) with a wide range of hosts like BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS and BL21(DE3) Codon Plus cell lines. But if you are using a modified MBP vector you have to consider some further optimization. You can find detailed protocol from the below link.
https://www.neb.com/~/media/Catalog/All-Products/FF91B67397D945E0956F3483174CF7E3/Datacards%20or%20Manuals/manualE8200.pdf
Dear Mahesh,
I have personally used the NEB Express cells and they work fine.
Abhishek
Have not noticed dependence on strain when expressing MBP-tagged proteins.
Dear Mahesh,
Using E.coli strains for the expression of a gene is a matter of optimisation. Different strains expresses the same gene differently. I am using specifically two strains for expressing my genes namely BL21 (DE3) and JM109 and the vector I use for cloning is pET28a(+)... I got a good overexpression using BL21(DE3) and that too in soluble fraction... So, you will have to check which one expresses your gene properly and where you get your protein ( in soluble fraction or as inclusion bodies).
All the best Mahesh.
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