I am having trouble expressing highly positive proteins in Bl21(+) cells. All of the proteins have pI values above 9 and none of them seem to be expressing. I am trying one inner membrane protein and two soluble proteins in the periplasm. Any suggestions on any other type of cells I can use or any other method I can carry out to express these prteins
Are your proteins not expressed at all, expressed at low levels, or expressed but insoluble?
If you have a purification rather than expression problem then you could try increasing the salt concentration of your buffers. I found that purifying proteins with high pI's worked best for me in buffers with high ionic strength (300 to 500 mM NaCl), as these proteins precipitated quickly when I used buffers with less than 300 mM NaCl during the first few purification steps. This makes sense as these proteins are obviously highly charged
I successfully expressed several viral nucleocapsid proteins in Bl21 Codon+ and Rosetta cells. Nucleocapsid proteins have a strong positive charge (pI > 9) as they have to bind very tightly to their viral genomic nucleic acids (their main job is to protect the nucleic acid from damage by the host at all cost).
As Wei mentioned, adding a solubilisation tag might help as well. We used pET28 and pET28-SUMO as our main expression vectors and the proteins expressed well with and without an N-terminal SUMO-tag (the tag was added to aid with solubility rather than expression levels). I used to grow the cells to OD600 = 0.6 at 37oC and then expressed the proteins at low temperature for a relatively long time (14 - 16oC for 48 - 72 hrs) with 1 mM IPTG. At higher temperatures a good proportion of the expressed protein was insoluble.
Depending on the pH of your buffer used for lysis, these proteins (if you have been successful in expression) could also be hitching a ride with negatively-charged cell debris components in your very first centrifugation step. Try having a high salt concentration during lysis and/or increasing the pH of your lysis buffer to 8 or even 8.5, to minimise the charge of the species you are working on. Hope this helps, Dave
Which vector did you use for your experiments? I think that BL21 cell is proper to pET vector series because of T7 promoter in pET vector and T7 RNA polymerase gene in BL21 cell.
I agree with Antonio.You'd better double check whether it's a purification problem or not. If the proteins couldn't be expressed at all, you can try to use expression vectors with large soluble protein tag(GST, MBP, SUMO etc) as above suggestion.
In additon, if the proteins are eukaryotic, you may try Yeast expression system or insect cell system, or mammalian cell system.
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