Hello to everyone,
We are study a recombinant protein (Escherichia coli) which has a huge hydrofobic cabity in its structure. By Electron microscopy we saw a extra density in this cavity and we decided to analyzed the content by Maldi-Tof. The result is in this cavity there are 3 differents types of molecules: Wax esters, phosphatidylcholine and sphingomyelin.
The questions are:
1) Is these molecules an usual contamination of recombinant proteins when we analyzed the samples by Maldi?
2) Which is the procedence of theses molecules? because they are present on animal cells and not in procariotes like E. coli?
We used a normal LB medium to express the protein ( nitrogenus bases, yeast extract and Sodium chloride) with E. coli DH5alpha (DE3). We extract the protein by cell press and purify in 2 steps: Ni-column and Gel filtration using as buffer: Tris 20mM pH: 7.4 and NaCl 150mM.
Many thanks!!!
David
PC is uncommon, but is found in bacterial membranes. How have you identified the lipid classes? Do you have fragmentation data as well as overall mass?
Have you tried expressing your recombinant protein growing E.coli in a defined medium instead of LB?
Yeast extract is a complex mixture and it might contain undesired contaminants that does not interfere normally with protein expression in E.coli but in your case it could be a potential source of artifacts in your protein ligand. Other sources can be your purifications columns, make sure to clean them properly because it might be another source of those lipids (I don't really think columns are the problem but it might be).
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