when we use bacteria as a host for produce enzyme and proteins after each bacteria Division ، gene of interest changes and generates a variety of proteins . How can i separate functional and nonfunctional enzyme or proteins for produce a pure and functional enzyme or protein?
You may use affinity purification to obtain an enzyme from a crude preparation but that doesn't guarantee an active enzyme/ protein unless it is interrogated for it's specific biological activity. For example cholesterol oxidases may be purified by using FAD or riboflavine-Sepharose column. So enzyme for which a cofactor is known can be easily purified. So you have to be specific for an enzyme oa protrin. Similarly Protein A column can be used for separation of IgG antibody from serum.
Once you have purified the protein, you can check it for mutations by measuring the mass using intact protein mass spectrometry, which can be accurate to within 1 Da.
You can use a standardized enzyme assay to measure the specific activity of the preparation for comparison to other preparations.
Unless there is a major change in structure (such as oligomerization or aggregation, or proteolysis), you probably will not be able to separate active from inactive protein. Aggregated protein can be separated from unaggregated protein by gel filtration chromatography. Different oligomeric states can also be separated from each other this way.
If the enzyme requires a cofactor, maximize the occupancy of the cofactor by supplying it during or after the purification.
In terms of functionality, the best a quick approach is to measure its activity as previously suggested
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