Why?

Peter pxpst2 at vms.cis.pitt.edu
Sat Jun 26 19:51:53 EST 1999


In article <377341D5.F2A738D1 at nic.bmi.ac.cn>, wangqm at NIC.BMI.AC.CN (Zhang
Bo) wrote:

> We have expressed a gene in E.Coli, but its molecular weight is not
> correct. We use mass spectrum and find it has more 500 dalton than its
> putative sequence. After sequencing the protein, we find no incorrect
> residue in both end (first 16 residue in N-terminal and the last
> residue),comparing with the protein sequence. We purify it from
> inclusion body in E.coli.
> Who can tell me why?

What is the expected MW and is it a Eucaryotic protein?

The cursory answer is that post transltional modifications have occured. 
Also, Mass specs will give one problems if the protein of interest has
seen or come in cantact with acetic acid or any dehydrating agent.  If you
used Maldi, then you will not be able to confirm whether modifications
have occured.  What will nail down the sequence would be to do thermospray
MS MS.  this will involve the digestion of your protein with a protease
(ie V8) and the characterization of the fragments.

regards,
Peter



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