[Nescafe]

Hi,

I read that to do NMR studies on 15N labelled proteins first we need to make sure that the protein can be 15N labelled which makes me ask the question what protein would not be able to?

Nescafe.

[Babcock_Hall]

Are you familiar with the overexpression of proteins in E. coli or other microorganisms?

[Nescafe]

Are you familiar with the overexpression of proteins in E. coli or other microorganisms?

Hi,

Do you mean using genetic engineering to incorporate the DNA sequence of a desired protein and expressing it in a fast growing organism? I am familiar with the general idea of how proteins are expressed in such a way to obtain a large quantities for in vitro studies etc.

Nescafe.

[Babcock_Hall]

There are at least two ways in which one supplies the nutrients for these genetically engineered bacteria.  One is called minimal medium, which consists of glucose, ammonium chloride, phosphate, and some micronutrients.  The other is called defined medium, which consists of the twenty amino acids, bases (components of RNA or DNA), vitamins, micronutrients, and phosphate.  I probably left out a few things, but I think I covered most of them.  Both of these media could be used to produce 15N-labeled proteins.  How would it be done, and how would the results be different between the two media?

[Babcock_Hall]

Are you familiar with the overexpression of proteins in E. coli or other microorganisms?

Hi,

Do you mean using genetic engineering to incorporate the DNA sequence of a desired protein and expressing it in a fast growing organism? I am familiar with the general idea of how proteins are expressed in such a way to obtain a large quantities for in vitro studies etc.

Nescafe.

From a cost standpoint, having good overexpression of a protein is very important in a labeling experiment.  With respect to your initial question, if one cannot obtain a protein from an organism that has can be fed some source of N-15 cannot be labeled.

[Nescafe]

What kind of protein would not be able to do such a thing? I mean lets say instead of ammonium chloride in the buffer we use 15N labelled ammonium chloride. Why would it not pick it up during construction of the desired protein?

I am just thinking of reasons or proteins that would not do this :/

Thank you for the help though really appreciate it =)


Nescafe.

[Yggdrasil]

While bacteria are capable of growing in media containing ammonium chloride as its only source of nitrogen, other organisms are not (for example, insect cells, mammalian cells).   So, if your protein cannot be recombinantly expressed in bacteria, it is much more difficult and expensive to obtain labeled protein.  Some reasons why proteins may not be amenable to bacterial overexpression are: overexpression of the protein is toxic to the bacteria, bacteria lack the proper machinery to process the protein correctly (e.g. various forms of post-translational modification like glycosylation), or the protein just does not fold correctly in bacteria.