Hi,
We are trying to express and purify a bacterial lectin (mannan specific) in E. coli. After sonification, we would like to apply the crude extract to a mannan-agarose (Sigma Aldrich) column. However, we are having troubles eluting the protein out of the column...
So far, we have tried 20% ethanol. 75% ethanol, absolute ethylene glycol (which destroyed our resin...), 1M NaCl, 2M NaCl, 0.01M NaOH (pH 11) and 0.5M NaOH (pH around 12-13). Only 0.5M NaOH appeared to elute the protein out. Is ok to use elution buffer at such high pH? We are worried if the strong base would denature our protein.
After Googling around, we saw some threads claiming desalting out the strong base would immediately refold the protein to the correct fold and retain the activity. Is that true? Will the lectin activity recover once we remove the base?
And one last question, will the mannan-agarose resin tolerate the strong base? We could not find any information on that either.
BTW, just incase someone asks, we cannot elute with mannose, as the protein is mannan specific. And we are not considering yeast mannan either as it is way too expensive. So NaOH seems like our last option.
Thank you so much!
Hi, Peter,
Why don't use 1,3-diaminopropane to take sample from column? This is appropriate for some lectins we work on Galactose column? Moreover, NaCl caused some problems on our lectin from a plant. I suggest to not increase alkali more than 8 and use 1,3-diaminopropane. Unfortunately, there is no useful information on lectin purificatione xcept for its expression using cDNA synthesis and specific primers.
Best Wishes, A.
Hi Peter. We ran into a similar phenomenon with a chitin binding domain from the yeast Kluyveromyces lactis. When bound to chitin it was nearly impossible to elute. We found that brief exposure to NaOH would cause rapid and complete dissociation from chitin (see attached ref). We went on to use the domain as an affinity tag and we took to using that method for elution, but we were always worried, so we eluted directly into a buffer that would immediately neutralize the eluate (not shown in the study). For our protein, that worked fine...the protein stayed active..as did every protein we ever fused it to. I hope this info is of some help.
I cannot say off hand if your resin will tolerate the exposure to base...there is one way to find out ;)
Article Characterization of a Nucleus-Encoded Chitinase from the Yea...
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