Hi Saeideh,

more information as to how you have looked for expression your protein would be useful. Are you looking at westerns? Does it have any tags? Also which expression strains have you tried?

However from the information you have given I take it that you have cloned the gene in frame with the lacZa gene, which you are expressing from the lac promoter. It's worth double checking that your insert is in frame with the start of the lacZa, otherwise you won't get the expression you want.

Looking at the vector (it's not one I've used) it seems to be designed primarily as a cloning & mutagenesis vector with the lac promoter there mainly to enable blue/white selection using disruption of the LacZa. The vector is not one I'd recommend as a good, modern expression vector. I suggest you take a look at the pET system.

-Richard

Your question miss much information but here are some thoughts:

* Looking at the map pALTER-1 seem to have two promoters - P_lac and P_T7.

I understand you are trying to express from the P_lac. Why so? It is much weaker then the P_T7.

* Which E. coli do you use? the vector has no lacI gene so unless there is such a gene in the bacteria genome IPTG will have no effect.

* Finally, assuming all else is done properly did you check for inclusion bodies?

Yoram

you can try something very simple : culture in low temperature 28-20...

Thank you for all your advice. The reason I am expressing from the Lac promoter is because the bacterial cells I am transforming the plasmid into ( BW25113) do not express the T7 polymerase.  The cloned gene contains a secretion signal so to test for expression, I check the activty of the enzyme in the media with a substrate that upon cleavage releases PNP into the media changing the color to yellow.  I have ran a gel as well and the correct size band does not appear on the gel.  I have checked the inclusion bodies as well.

I am using this vector as I require a vector with Tet resistance since the BW25113 cells  I am using carry both Kan and Amp resistance genes. I have tried replacing the Kan cassette in Pet28a with Tet cassette, however given there are no restriction sites that will completely take out the Kan cassette, I have not been successful.

Any help in terms of expression from this vector or suggestions for a different vector is much appreciated.

Hi Saeideh,

some thoughts:

in E.coli proteins are not actively secreted into the media, with an appropriate signal sequence such as OmpA or PelB they are transported into the periplasmic space. So assaying the media is unlikely to be successful.

Your construct (if I've understood correctly) will be Met-LacZa(1-18)-ss-GOI-stop. Having the 18 amino acids before the signal sequence will severely compromise it's activity.

Do you have any specific reason for using BW25113? Choosing expression vector first then testing suitable expression strains would be the better way to go. Sometimes expression can be improved by using a different strain. It would also let you use a pET vector - which you correctly realise you can't with BW25113.

-Richard