I try to display an enzyme on the surface of E.coli cells. To do this I have made a chimeric protein consisting of signal peptide (SP) - enzyme (E) - outer membrane protein of E.coli (MP). The signal peptide works perfectly since more than 95% of SP is cleaved in SP-E chimeric protein under the conditions of induction of protein expression (induction in modified TB medium (YNB instead of yeast extract) at 37C with 1 mM IPTG during 2-3 hours). I can see expression of SP-E chimera, but not the full construct SP-E-MP. I have tried to express this consrtuct using BL21(pLysS)-pET system with T7 promoter and BL21(pLysS) - pBAD with arabinose controllable promoter. None of the systems works for whole chimeric protein. Since SP work well, the construct is transported into the periplasm but then failed to insert in the outer membrane, and perhaps is degraded. I suspect that conditions for expression and/or folding of membrane protein is far from optimal. Can you suggest a strategy to optimize conditions for expression of such chimeric membrane proteins? Which parameters of expression conditions are crucial? What kind of conditions have worked for your membrane protein?
This is never a simple thing to do. I would start by dropping temperature to 30 degrees to slow growth and allow more time for assembly. Are you seeing inclusions and are you separating the OM from Inner? I wonder if its getting stuck in the periplasm or your 1mM IPTG is overloading the ribosome - I would also look at lowering the IPTG. The fact that the arabinose is not working kind of precludes the former comment though but its food for thought. I would also closely look at my sequence to make sure my frames are in line and a frame-shift had not snuck in there to spoil the party. Like you say, you could indeed have folding problems and when doing this kind of experiment not everything is actually possible...good luck
I would certainly check the sequence of your construct. It is quite surprising that you don't see the full length protein. If protein translocation or OM-insertion kinetics are problematic, you should at least see it inclusion bodies containing the full length protein. At any rate, go down to 30°C, and use strains that slow down expression on top of that. pET even in pLysS is just a bit too much. Try Lemo21 instead. Or play around with your arabinose concentration using your pBAD construct. If your enzyme >30 kDa, you may want to consider a different system of display altogether, namely using an autotransporter as vehicle. There is no guarantee for secretion but if it works, results are stunning. Have a look at our recent paper in Microbial Cell Factories 06/2012; 11(1):85.
Simona, you can create the separate question. Each question is tagged by keywords. This way your question will be visible for many other researchers.
I have sequenced my constructs. Everything is fine. I tried to detect my protein by reducing SDS-PAAG in whole cell lysates and by assaying activity of the displayed enzyme. PAAG shows no visible difference before and after induction. However, there is a very weak enzyme's activity on cells expressing in from pBAD.
My enzyme is
The protein does express, but little. You could make a fractionation of E Coli to find out where it is, to get some idea if traffficking is fine or not.
What is the OM-protein ? Did you check whether the topology of the fusion is correct with respect to the native topology of the OM-p ?
From preliminary experiments I know that the signal peptide works perfectly (> 95% is processed). Therefore, I think my protein goes to periplasm. Then only a part of it correctly folds and inserts into OM. My OM-p is one of the E.coli adhesins and belongs to the family of autotransporters. The passenger of this protein is changed for my protein and native linker is preserved (that is crucial for display). C-terminus of the OM-p is not modified.
I suggest that due to overexpression, the OM-p titrates out not only the signal peptide processing machinery but also chaperones. As a result, it folds incorrectly and rapidly degrades by proteinases. Today I have found conditions that elevates the level of correctly inserting and functioning chimeric protein. These conditions are low temperature and low concentration of inducer. They provide a slow production of the protein, so it has time to fold and insert into OM. I can not see the protein by PAAG, but activity of my enzyme displayed on the surface of the E.coli cells has increased greatly. I think this is good conditions for expression, but they can be still improved.
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