I am looking for an example of a certain protein:
It should meet the following criteria:
- it is an enzyme
- it can be expressed heterologously in Escherichia coli in the cytoplasm (ideally shown before)
- There is an easy, established, spectrophotometric assay (ideally continuous, something in the UV range like NAD)
- the assay for it doesn't show activity (or very low) in wild-type E.coli (means E.coli doesn't have the gene or substrate doesn't react with E.coli version) - this is where I have the biggest problems!
- ideally the activity has no issues with Bugbuster/BPer cell lysis
- not toxic to E.coli
Who has an idea how to look for something like that?
Are there search engines that help me find that? Or does anybody have good examples or such proteins?
Dear Johannes
bacterial sortases A or sortases C may be a possible candidates for you.
My experience is restricted to GBS sortases C
references:
Article Noncanonical sortase-mediated assembly of pilus type 2b in g...
Article Group B Streptococcus pilus sortase regulation: A single mut...
Article Structure analysis and site-directed mutagenesis of defined ...
those can be easily expressed in high yields in E.coli and purification using BPER or Cellytic express do not affect their activity and their activity could be easly monitored using EDAN-DABSYL quenced fluorescent peptides.
Those are very specific enzymes belonging to gram positive bacteria and i do not think that in E.coli you can found nothing that cross-react.
good luck
Manuele
How about GST? It expresses so well that it can be used as a tag to improve the expression of other proteins. You will express so much of it that the contribution of endogenous GST will be negligible. (you can see the ratios by purifying something with a GST affinity resin -- endogenous GST simply doesn't show up). You can buy assay kits for activity.
There are probably thousands of examples. First of all it does not need to be heterologous, you could use an E. coli gene where the wild type is deleted from the genetic background, for example lacZ. Or if you want heterologous you could look at nitrilases, amylases, laccase, pectinase, amidase, catalase, invertase, the list goes on.
Dear Johannes,
I am working with cofactorless, ring-cleaving dioxygenases from different organisms, of which some are easy to express. The reaction is easy to follow continuously via UV/VIS-spectrophotometer in an assay that only needs buffer, substrate (relatively expensive), and enzyme. E. coli is not capable of cleaving this substrate.
If you are interested, check my papers :)
Best regards, Sandra
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