pEt-Expression-Mystery

Antonin Tutter atutter at aim.salk.edu
Sat Aug 8 02:47:56 EST 1998


In article <6q3sl4$dmt$1 at gwsun.medinf.mu-luebeck.de> , "Lars Komorowski"
<larskomo at physik.mu-luebeck.de> wrote:

>I am trying to overexpress a 17kDa-Protein in E.coli BL21 with Novagens
>pET24a Vector. I inocculate my LB medium from an overnight culture, let the
>culture grow until the OD reaches 0.6 and induce with 1 mM IPTG. After one
>night there is a band at 25 kDa in SDS-PAGE made with total cell protein.
>When I try to separate cytosolic proteins from the rest and make another
>SDS-PAGE with both fractions there is no band there.
>I have two questions:
>1. If the protein band is not the desired protein what can it be ?
>2. If the band represents the protein, is it possible that it is proteolysed
>within minutes ?
>
>
1)  You probably should experiment with induction times and induction
temperatures.  Even when I express very stable proteins using the pET system
in BL-21 cells, I only induce for 3 hours maximum.  You may even try 1.5
hours at 30 degrees.

2)  Some proteins migrate differently than expected.  Ususally highly
cationic peptides will migrate slower in the gel, appearing as higher
molecular weight.  This variance in apparent MW can be as much as 50%.

3)  Has this protein been successfully expressed before?  Have you verified
the sequence?



_______________________________________
Antonin Tutter
Salk Institute for Biological Studies
RBIO-J
10010 N. Torrey Pines Rd.
La Jolla, CA  92037
email:  atutter at aim.salk.edu
web:  http://www-biology.ucsd.edu/~atutter/




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