The enzyme has been produced as inclusion bodies and during purification it's activity was lost.
Hi. At what point in the purification procedure do you lose activity?
Do you see activity in cell extract? it is strange if you have your protein in inclusion bodies...How do you refold it? do you have a tag? sometimes you can see it soluble, but maybe is not properly folded...
Dear Steingrimur the enzyme activity is lost during 4 steps of chromatography. In brief urea is used for dissolving of IBs, then refolding is done on gel filtration column, nucleic acids are removed by ion exchange and then affinity chr. is used for final purification up to 95%. Dear Valentina yeah! It is seen in inclusion bodies which is being refolded by gel filtration chr. The recombinant enzyme has a His tag.
My question is that if there any procedure for retaining of enzyme activity in final preparation?
Hi Atousa. You need to be patient and identify the problem.
Where in the 4 step procedure do you lose most of the activity? Are you losing the enzyme activity or are you losing the protein?
Hi Steingrimur. You are right. We should find the problem. But now I have a purified enzyme (up to 95%) with half of expected activity! Is there any procedure for this preparation or I have to ignore it and prepare another one?
When you say that the prep. has half of the activity, are you talking about specific activity (activity/mg protein)?
Losing specific activity likely means that the enzyme is denaturing after the affy. step. Are you continually losing specific activity on your purified enzyme with time, or is there no change?
Losing specific activity with time could mean that the enzyme is not stable in your storage buffer. Losing specific activity only after the affy. step could mean that your elution conditions are too harsh.
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