peptide expression in E.coli

Wayne R. Baker baker at iastate.edu
Thu Mar 23 16:03:14 EST 1995


In bionet.molbio.proteins,   <M.R.douglas at rheuma.bham.ac.uk> wrote
:I am interested in producing peptides (10-30 a.a. in length) in E.coli with a
:view to labelling them isotopically for NMR experiments.  Does anyone out
:there have any hints, tips or references as to the best way to go about this?
:And which is the best purification method for sorting out the good from
:the bad? Getting them synthesised in the old fashioned way would break
:the bank, I'm afraid.

I haven't done anything that small, but we routinely label our 134 aa
protein with 15-N. All you need is a strain that grows in minimal media
and an expression system that gives you decent yields (we like to see at
least 30 mg pure protein from a 5L culture in minimal media). All you do
is substitute 15-N ammonium chloride for the non-labeled ammonium
chloride. You can also use 13-C labeled glucose but that's an order of
magnitude higher in cost. BTW, we have modified our M9 to include some
vitamins and minerals. We get happier bacteria and better yields. Let me
know if you'd like our recipe. 

As for purifying it, you'll need to figure out some protocol that is
specific for your protein. I assume that when you say, "sorting out the
good from the bad," you mean separating labeled from unlabeled. I'm not
aware of a way to separate an isotope-labeled protein from one that
isn't. However, using 95% + enriched 15-N ammonium chloride media will
get you a 95% + labeled protein since it's the sole N source for the
bacteria. 

BTW, I tried e-mailing you but it bounced. :-(

Wayne Baker (baker at iastate.edu)         Few things are harder to put up with
4288 Molecular Biology Building         than the annoyance of a good example.
Iowa State University                   -Mark Twain
Ames IA 50011                   
  (515) 294 0781   



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