2 replies to this topic

[donny]

I have cloned the gene for a membrane protein along with the pre-gene sequence including the RBS into a low copy number vector (~5 copy/cell) that is a derivative of pUC19. However, when I transform the plasmid into cells with the gene deleted, the activity of the membrane protein seems to be absent. This contradicts the results in literature. Although I cloned the gene slightly differently, I cannot figure out why my method would not work.

To clone the gene, I designed forward and reverse primers to amplify the gene from the genomic DNA. Because the MCS is part of the LacZa gene (for blue/white screening), my forward primer has a stop codon to prevent augmentation of a small peptide to my membrane protein. This is then followed by the pre-gene sequence (22bp), which includes the RBS (AGGAGC).

LacZa in pUC19 multiple cloning site:
____________________ATG......GAGGATCCC.....
____________________M........E__D__P....
fwd primer:_______________NNNNNGGATCCCTAA......AGGAGC.....ATG
cloned plasmid:_____ATG......GAGGATCCCTAA......AGGAGC.....ATG(membrane protein)TAA
____________________M........E__D__P__stp_________________M__(membrane protein)stp

As illustrated, the fwd primer has the BamHI site. Following the site, a "C" was added before the stop codon TAA to be in frame. Then, the 22bp pre-gene sequence with the RBS is added before the ATG for my gene. I have sequenced the cloned gene in the plasmid and everything looks ok.

From my understanding, the lac promoter will allow transcription of an mRNA from the LacZa gene all the way to the end of my gene. However, two proteins will be translated. One is a truncated LacZa due to the added TAA. The other is my membrane protein, which has its own RBS. Is my understanding correct? Am I doing anything wrongly to prevent the transcription/translation of my gene?

Strangely, the paper that described the cloning of this gene did not even bother to add a stop codon like I did or make sure the gene was in frame. They did include the RBS though. Wouldn't this lead to the expression of a huge useless protein? Or would the included RBS ensure that the target protein is expressed in the correct ORF?

Please help!

Edited by donny, 26 October 2010 - 08:10 AM.

[protolder]

Hola, Im not sure that the signal peptide of an eucariotic protein is recognised by E. coli. If it ´s published it does, by in other time membrane and secretion proteins were cloned under bacterial signal peptides as The OMP-A in the Pin vectors. Good luck

[donny]

The membrane protein is actually an E coli protein but I've only knocked out the gene and cloned the gene into a plasmid.