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No expression of 7kDa His-tagged protein - why? - Poor protein expression (Jun/22/2005 )

I have cloned a 97-bp sequence into pTrcHis, a HIS-tag vector. The sequence was optimized for E. coli codons. The plasmid was transformed into E. coli DH5aMCR. The clone was confirmed by sequencing, so is in-frame. The protein produced should be 7kDa (including the His-tag). Induction is performed by growing the cells in LB-Ampcillin from OD600=0.03 to OD600=0.5 (~3 hours) and inducing with 2.5mM IPTG for 3 hours at 37oC. I run samples of whole cells on 15% SDS-PAGE and stain with InVision (His-tag stain) and Coomassie blue. I also did a TALON resin purification on 500ml of cells. In no case do I see or purify any protein.

I have cloned and purified over 30 different proteins using this method and have never failed to, at least, detect the protein in an SDS-PAGE gel. My question is... does anyone have any thoughts on whether the small size of the protein (7kDa) has something to do with the apparent lack of expression? All the other proteins I have worked with have been between 28-150kDa. Are there special considerations that have to be taken when growing cells and expressing small proteins?

Thanks.

-BHJ-

hi, probably my question is silly, is there any chance that your protein got secreted?

-dtle-

Are you sure you didn't run your protein off the gel? 7kB is mighty small.

-pBluescript-

QUOTE (dtle @ Jun 22 2005, 05:58 PM)
hi, probably my question is silly, is there any chance that your protein got secreted?


No. I run 15% gels and I have a 7kDa "marker" protein. Also, using SignalP, it does not predict a signal sequence, so I doubt that the protein is secreted.

Also, I sequenced through the all the regulatory elements, and the promoters, enhancers, etc... are all intact.

Good ideas though! Any other thoughts?

Thanks

-BHJ-

Is this 7kD fragment part of a transmembrane domain?

We failed miserably several times to get protein production in a prokaryotic expression system only to realize later that elements of the protein were part of a transmembrane domain.

-pBluescript-

QUOTE (pBluescript @ Jun 23 2005, 11:59 AM)
Is this 7kD fragment part of a transmembrane domain? 

We failed miserably several times to get protein production in a prokaryotic expression system only to realize later that elements of the protein were part of a transmembrane domain.


I don't know if it is or not. The peptide is from a eukaryotic protein of unknown function. I usually work with bacterial proteins. Is there a program to determine this?

Thanks

-BHJ-

Is there any way you can shift up or down the sequence a kb or two. Yah I know you have to reclone and all..

-pBluescript-

Hi BHJ,

How's about fusing your protein with anther protein for expression? for example fuse to GST.

-dtle-

QUOTE (dtle @ Jun 23 2005, 07:15 PM)
How's about fusing your protein with anther protein for expression? for example fuse to GST.


I have somewhat come to the conclusion, that this protein is being targeted for proteolysis for some reason. You are right, I think it will have to be attached to a bigger tag, like GST.

Thanks for your help.

-BHJ-

I have somewhat come to the conclusion, that this protein is being targeted for proteolysis for some reason. You are right, I think it will have to be attached to a bigger tag, like GST.

Thanks for your help.
[/quote]


Are you sure your protein is degradeted? why do you think so? I have the same problem you had - I have 7 KDa protein and i canĀ“t see it on gel using coomasie either after blotting and staining with Ni-peroxidase konjugate...

-Bobikpp-