I would like to achieve heterologous overexpression of a mycobacterial enzyme in E coli that harbors a Fe-S cluster. Unfortunately, the expressed and purified His-tagged protein apparently lacked Fe judged by UV-VIs and also the inherent activity was also missing (not so much surprisingly, then). What would you recommend for promoting the expression of the holo enzyme? In fact, the FeCl3 supplement in the LB medium was also tested but was not successful.
Many thanks for the comments and suggestions!
Gergely
You may be able to get some useful ideas for improving the situation from reading papers such as this one (and references within) about optimization of expression of Fe-S proteins in E. coli.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2080846/
Fe-S clusters are (nearly always) VERY sensitive to oxygen. The first thing I would try is to repeat the expression under anaerobic conditions. E. coli will grow slower but still you can often obtain high quality holo protein this way. If this does not work, I would try co-expressing with a GroEL/ES chaperonin plasmid and/or along with a plasmid that overexpresses the Suf or Isc proteins (iron-sulfur cluster assembly, etc). These can be obtained from a number of academic groups upon request. If all of this fails, I recommend deliberately expressing the apo protein, then refolding in vitro. This can sometimes work if you have a single domain protein (often fails for multi-domain proteins).
One basic question: Are you certain that sufficient iron is bio-available? Past experience with high density E, coli fermentation showed that siderophore production was quite significant as evidenced by the appearance of a red-pink chromogenic substance during the isolation of the recombinant protein. I identified the substance as a siderophore which is produced when microbial organisms are iron starved even though inorganic iron (perhaps not bio-available) is in the media. Changing the iron source and amount resolved the issue and increased the expression level and cell density as well. Siderophores have a shockingly high affinity constant for iron and humans and animals stand no chance in competing with microbes for iron.
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