Many proteins are oxygen-sensitive. If I want a lot of those proteins, is there any solution to express and isolate those proteins without loss of their activity?
Need to eliminate out oxygen in the system by following either one of the following:
1. Deoxygenation by allowing high purity deoxygenated nitrogen gas to pass through the system.
2, Introduction of sufficient amount of any antioxidant that will not interfere on the activity of the enzyme of interest,
for small scale, I used to degass the water by passing argon or nitrogen gas through it as it was cooling after boiling. Then add sodium dithionite. DTT is not good enough, typically. Then grow the bacteria in carboys with tops. If you have a glove box, you can purify and assay in there. Otherwise you may need to purify outside the glove box and possibly reactivate your enzyme. I used glass capillary type tubes for the assays and sealed the ends with a flame. During the purification degassed water, with dithionote was used in the buffer. And when I ran a column, I collected the tubes and passed argon over the top before sealing them with rubber stoppers. I never had a glove box. My enzyme was lysine 2,3 amino mutase from clostridia. For reactivation, it also required dihydtolipoamide. You can check out my publications for details.
By far the best solution to this is to use an anaerobic glove box to break open the cells and purify the proteins of interest. This requires quite a lot of investment (dedicated glove box and FPLC system inside it). We have used this approach to study many O2-sensitive proteins, including the O2-sensing transcriptional regulator FNR from E. coli.
Someone mentioned adding Sodium dithionite. Do I need to add it during cultivation or during protein purification? What is a good concentration to use?
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