Hi,

I am trying to express an eukaryotic membrane protein (only ectodomain, 60kDa, pI 5.6) in BL21DE3. I have played around with IPTG con. and temperature of induction to get the protein in soluble form but was not successful. So decided to extract the protein from inclusion bodies and refold it. I know that other bacterial host like Rosetta, pLys or origami would have been a better choice. Even at 0.05mM whole protein was found in pellet only. With higher con. of IPTG protein aggregation was observed after lysis (cloudy/ flakes) (lysozyme + Dnase + sonication (amplitude 20, 10s on , 40s off total time 5min). At 0.05mM IPTG induction, the pellet after lysis has two layers. An upper yellow translucent layer (L1) and lower white layer (L2). As a control, I have transformed BL21DE3 with the other plasmid (xy) which express the protein as inclusion bodies. The pellet from these two expression vectors were different. The control (xy) gave complete translucent yellow pellet unlike my construct. I seperated the two layers (pellet of y protein) and found that my protein is found entirely in white bottom layer (L2). When I tried to solubilize the protein in buffer using pellet (L2) (8M Urea, Tris 25mM, pH 8) very little protein got solubilized. I came across an article which suggested to use alkaline pH for solubilization. Upon adjusting the pH to 13, I got lot of solubilized protein even at 2M Urea. As the pH was very alkaline (13) (Ni-NTA/Talon doesn't work above pH 9) the pH was lowered to 7.4 by adding HCl. Precipitation was not observed. But the problem is that when I tried to bind the protein (solubilized in 2M Urea) to Ni-NTA/Talon (used both) there was a little binding to the beads as most of the protein was retained in flow through. I repeated the experiment with 8M Urea as above, yet no difference in result. My protein has His tag at N and C terminal confirmed by western blot using anit-his anitbody. I checked the beads with other his tag protein (beads are working fine). I tried many additives like glycerol 10%, KCl 100mM, B-Mercatoethanol 10mM, DTT 5mM (I know Ni-NTA and Talon wont work at higher con. of DTT, usually it works fine at this con. as it did many times in my hand, also I replaced it with Mercaptoetahnol).

My questions are

1. Is the white part of the pellet is an aggregate? Does protein forms aggregate in inclusion bodies?. Is it mandatory to avoid it. If so how?

2. When the protein got solubilized, why it is not binding to the Ni-NTA/Talon beads as I can see that the elution pattern looks similar to the before binding (I have also avoided using EDTA and DTT).

3. How to avoid precipitation during dialysis. ( I tried to remove urea very slowly by continuous dialysis method which takes 5 days to remove urea).

Regards