peptide expression in E.coli

[MARCUS, DR J.]

>To:            proteins at net.bio.net
>From:          M.R.douglas at rheuma.bham.ac.uk
>Subject:       peptide expression in E.coli
>Date:          22 Mar 1995 09:54:59 GMT

>M.R.douglas 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.

>Here's hoping!

>Mike.

I Have not done what you are talking about but I have 
considered it.  You might think about the following:

- Some of the isotopic media reagents you would be using 
are quite expensive themselves so you should really limit
the volume you grow your bugs in.

- You will really want bugs that express your protein at 
the highest levels possible so as not to waste your label.

- I think you should consider mutant E.coli that are 
lacking in particular pathway(s) for amino acid biosynthesis. 
This aught to help you attain the highest incorporation of 
label into your protein of interest (if you wanted your 
label incorporated into a specific residue).  Of course, you will 
need a defined media to grow your bacteria in.

- Since your peptides are so small, you might be able to 
use a size exclusion step to get rid of many of your larger 
proteins.  Or maybe an Ultrafiltration membrane with a M.W. 
cutoff of say 10,000.

I am sure you have a few more things to consider but I will 
leave it there.  

Regards,
John Marcus
John Marcus            Marcus at tpp.uq.oz.au (Dr J.Marcus)
Cooperative Research Centre for Tropical Plant Pathology
5th Level John Hines Building
University of Queensland
St. Lucia, QLD 4072
AUSTRALIA
Fax: 61-7-365-4771
Phone: 61-7-365-4764