What is the best composition of lysis buffer for E.coli cells carrying recombinant proteins? How can we increase the efficiency of lysis buffer to enhance downstream processing of the proteins?
Thank you. But I want to purify the protein through Ni-NTA column as my protein contains his tag. Presence of EDTA in lysis buffer will affect the binding.
I agree with Prabha's answer. However, I wouldn't care too much about the buffer composition provided that it suits with your protein of interest in terms of pH and salts (stability and solubilisation of your protein and compatibility with downstream purification) and that it contains proteases inhibitors and benzonase (or DNase + RNase). Concerning lysis efficiency I use glass beads / vortex to brake E.coli cells for my protein productions (2 liter flask culture scale) and the yield is always good. Please feel free to ask me the procedure : I will send it to you.
You can use any buffer(pH) and salt combination in which your protein feel good (stable and soluble). stability can be decided after purification only so at this stage solubility is the main concern. According to the pI of the protein you can select best buffer combination.
If you're immediately going to be purifying by Ni affinity, then I would use a buffer composition similar to 20 mM Tris-Cl pH 8.0 with some imidazole added (~20-50 mM). That's the suggested binding conditions for commercial Histrap FF columns and I've used it successfully in the past with other, non GE products as well.
It's pretty well established that adding imidazole in your binding buffer dramatically improves purity of your desired protein. Just be aware that not all recombinant, polyhistidine tagged proteins bind well to Ni-NTA, presumably because the poly-his is partially embedded in the protein and therefore adding imidazole may negatively affect your yields.
If this buffer condition is not suitable for your downstream purposes, then I would suggest seeing if your protein is stable in it first by extracting in to these buffer, letting it sit for an established time (approximately how long it would take to purify the protein by Ni-NTA in your hands) then exchange the buffer in to your optimized activity buffer condition and assay activity. If it maintained activity, then use my suggested conditions. If not, then you'll likely have to do some old school biochemistry and simply try a number of buffer conditions in a systematic fashion.
I usually use Tris 20mM, NaCl 500mM, Imidazole 10mM pH=8 for the lysis of E.coli containing recombinant protein with 6xHis tag.
- Badges
- Science method