I have expressed my recombinant protein in E.Coli with a N-terminal his-tag. Cell lysis was done with BugBuster, purified successfully with IMAC (Co-resin) and the immidazole removed through a buffer exchange (so as to not interfere with absorbance readings during the enzyme assay). Activity is definitely reflected in Whole cell extracts without BugBuster lysis (sonication used instead), but with purified BugBuster-ed protein, there was no activity recorded with the enzyme assay.
Any thoughts or possible reasons as to why?
I suppose it means one of the secret ingredients in the bugbuster must be inhibiting your enzyme. Since it's secret, we can't easily know what it might be. The base is mild detergents, which should not normally be a problem (unless your protein is funny...). Then there is some unusual branched compound that makes the initial break in the cell membrane. It may or may not have something to do with it.
You probably shouldn't be overpaying for this so-called product. Look up the STET / lysozyme /dnase method, from a source like Maniatis. That uses a single freeze-thaw cycle to make the initial dent in the membrane. It's basically the same as Bugbuster, minus the (senseless) secret sauce, and minus the (insane) price tag. If that's still inactive, then you know for sure that your protein is unusually vulnerable to common lab detergents.
Perhaps one or more of the detergents in the Bugbuster mix denatures your enzyme.
Dear Sarah
is it certamilly possible that the non. ionic detergent present in the buffer can affect the protein folding and activity if the protein is not very stable. but dis you know if you obt in active protein with a different lysis approach as sonication or French press that do not require detergents?
because if not, you need to consider also other factors; for example:
some times proteins are not active after purification also using sonication because miss some cofactors which are not present in e.coli or because lost it during purification.
for example zinc proteases may bind nickel from the coloumn and you need to remove it by edta and add the zinc to make it active.
one other doubt: is your enzime contain cisteines? are you sure that it reduction or oxidation state is it correct because if it contain cisteines involved in s-s is possible that are not formed, protein is not folded and not active but this is not a bug buster fault.
if the cisteines are catalytic is possible that are oxidated and you need to reduce it by dtt or tcep to activate your enzime.
good luck
Mamuele
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