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Problem with protein expression

Problem with protein expression - Protocols and Methods Forum

Problem with protein expression - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 09-29-2004, 01:38 PM
Peter Frank
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Default Problem with protein expression



Hi,

I have some problems expressing a His-tagged protein. I cloned the
sequence in-frame into the pET-20b(+) expression vector, which I used
to transform E.coli BL21 (DE3) cells with. I thoroughly checked that
insert and His-tag are in-frame. I grew the culture at 30 C, and
although it grew slowly, it did grow continuously as monitored by OD
measurements. So, I assume the protein to be expressed is non-toxic. I
lysed the cells completely. I purified some lysate using Ni-NTA
magnetic agarose beads, which should highly enrich the His-tagged
protein. I ran several SDS-PAGE gels with purified and unpurified
lysates (with non-reducing and reducing sample buffer). The
silver-stained gel did not show any band with significantly increased
intensity (but then again there were many other bands of course that
may obscure this).
Two other gels were immunoblotted and a) incubated with an antibody
against a peptide segment of the protein to be expressed as well as b)
incubated with an anti-His-tag antibody. Western blot a) showed faint
bands of the appropriate size whereas Western blot b) showed
absolutely no bands for the purified lysates and bands of the wrong
size for the unpurified lysates (too low MW).
This is very confusing and I am not sure where the problem could be.
The expression vector seems to be OK, but I cannot detect a His-tagged
protein of the expected size using anti-His-tag antibody.

Does anyone have any idea where the problem could be or can you
recommend a good troubleshooting guide for prokaryotic protein
expression?


Peter
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Old 09-30-2004, 12:08 PM
Kyle Legate
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Default Problem with protein expression

Peter Frank wrote:
This may be a dumb question, but did you induce your cells? You didn't
mention this step. There is an E. coli protein that reacts with the anti-His
antibody (at around 60 kDa?), and you may be detecting this in your Western.

It's a good idea to compare some induced E. coli lysed directly with SDS
loading buffer with the lysate you intend to use for purification to
determine if your protein is soluble. Garbage in gives garbage out.

The Qiagen Ni-NTA matrix product manual is fairly comprehensive for this
type of purification. It should be available as a pdf from their site.


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  #3  
Old 09-30-2004, 08:02 PM
Peter Frank
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Default Problem with protein expression

Kyle Legate wrote:


Forgot to mention it. I did induce my cells with IPTG at a final
concentration of 0.4 mM.


The band I see is around 30 kDa. Don't know what it is.


Does SDS loading buffer lyse absolutely everything?


Peter
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