my primers amplify a region containing signal peptide also. this fragment was cloned into pet28 vector and expressed in BL21 expression host. My protein band was not expressed initially and when expressed was not able to purify by Ni-NTA column method as the protein didnt bound to coloumn under both native and denaturing conditions
Hi, Did this lineage able to grow after induction?
I am asking that because I did the mistake of do not take off a signal peptide from that protein I was trying to express in E. coli. The point is: Yes, this signal peptide can influence if this protein will be toxic meaning that the cell cold not be able to grow or if the amounts of protein will be very low alter induction. I solved that by taking off the signal peptide.
Are you expressing in E.coli an eukaryotic protein? Often after signal peptides there is a protease digestion site. If your construct is correct by sequencing, start your protein production from colonies deriving from the original clone streaks on LB-agar plate. Run a western blot with cell lysates using Mab anti-His(H1029 Sigma ) and compare the signal with a cell lysate from E.coli pet 28 empty. If you will get black signal your protein is there, you have only to set the right conditions. Write again and let us know :-)
Are you trying to get periplasmic expression? There doesn't seem to be a need for the leader sequence in E. coli unless it helps to increase expression level and can be cleaved off later. It's not involved in folding like pre or prepro domains sometimes can do. Usually these leader sequences contain unpaired cysteines that can interfere with, intracellular location, folding, and purification. Is the His tag therefore on the N-terminus?
I suspect the expression level is low in which case the host cell contaminants are quite effective in competing with the His tag due to the presence of His motifs. Are you using the Qiagen Ni-NTA resins and being cautious about the number of interfering substances? Are the codons optimized for E. coli preference particularly at the 5' end? When you check with anti-His antibody you might want to do this as a Western blot; what would you expect if the protein were clipped resulting in a fragment harboring the His tag?
Yes, a signal peptide has a big influence on 1) synthesis of the recombinant protein and 2) stability of the construct in E.coli.
If the gene of interest is eukaryotic, then the protein probably requires the folding chaperones and oxidising environment of the endoplasmic reticulum to fold properly.
If you express it without the signal peptide in E.coli, it may have problems folding in the cytosol (which is reducing). If the protein is normally glycosylated, then that won't happen either, so there could be a lot of problems, but you could also be lucky.
If you clone the coding region with the signal peptide into your E.coli expression vector, then you may have other problems. Translocation across the ER membrane has evolved from translocation across the plasma membrane in prokaryotes, but it is not exactly the same any longer. Bacterial translocation pores are more demanding with respect to signal peptides and mature peptides following. Some eukaryotic genes with signal peptides will jam the E.coli translocation pores at the plasma membrane and they are toxic to E.coli. You can spot this if you notice that that correct recombinant clones grow slower than those containing empty vector. If that is the case, you can try to replace the eukaryotic signal peptide by a prokaryotic signal peptide, or directly clone just the mature portion of your coding region of interest into an E,coli expression vector designed for periplasmic expression (it has the polylinker after a prokaryotic signal peptide, often from Erwinia pectin degrading enzymes). The E.coli periplasm is also oxidising, like the endoplasmic reticulum lumen of eukaryotes, and you can get disulphide bonds etc... but if your protein is normally glycosylated, then you can't use E.coli if you need the glycans. Then try expression in insect cells or yeast.
Good luck :)
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