I am working on cutinase gene and this gene generally shows extracellular expression. So, that means I should have my protein in the media. I am using BL21 E.Coli as a host expression system. How I can purify and concentrate this protein for SDS PAGE run. I have many cutinases to express, so it would be better to follow a protocol which is cost effective also.
If the proteins have an affinity tag, such as a His tag, then you can collect the protein from the medium by pumping the medium over a 5-ml HisTrap column at 20 ml/min. Then wash the column and elute with an imidazole gradient to purify the His-tagged protein. The same column can be reused for each of the cutinases.
If there is no affinity tag, then you can use a HiTrap ion-exchange column and a salt gradient instead. The resulting protein may not be as pure as with an affinity tag.
If the eluted protein is too dilute, the you can concentrate it with a centrifugal ultrafiltration device, or rebind it to the same column and elute it with a single step wash of imidazole or salt.
If the proteins have an affinity tag, such as a His tag, then you can collect the protein from the medium by pumping the medium over a 5-ml HisTrap column at 20 ml/min. Then wash the column and elute with an imidazole gradient to purify the His-tagged protein. The same column can be reused for each of the cutinases.
If there is no affinity tag, then you can use a HiTrap ion-exchange column and a salt gradient instead. The resulting protein may not be as pure as with an affinity tag.
If the eluted protein is too dilute, the you can concentrate it with a centrifugal ultrafiltration device, or rebind it to the same column and elute it with a single step wash of imidazole or salt.
Try ammonium sulfate precipitation. It is a cheap, easy, fast and robust way to capture protein.
From your profile, I assume that your cutinase genes are from plants. In order for the protein to be secreted, you will need to use a vector that provides a signal peptide that is functional in E. coli - I wouldn't think that plant derived sequences would work. You can also use the minimal medium recipe described by Bill Studier to minimize contaminating proteins. Just be careful to maintain healthy growth conditions (a dark zone in the cell pellet indicates dead cells that could shed protein into the medium).
Adam's suggestion of His-tag is quite good. The tag is small and usually does not need to be removed. You can't put it at the very N-terminus though because you need a signal peptide. The binding matrix is relatively cheap compared to alternatives, reusable as it washes quite clean, and can even be easily recharged with nickel or cobalt when necessary. No fancy equipment needed - you can even use gravity feed to pump the medium over the column. Or maybe even batch mode if you are doing very large volumes? - put the resin in the cleared medium to bind cutinase, pellet, wash, and put resin in column for final elution.
Article Stable Expression Clones and Auto-Induction for Protein Prod...
As such E. coli is a very stringent system.It does not secrete protein in extracellular medium unless it is coupled with extracellular targeting system such as tat. If you are trying to look for your over expressed protein without any such system there is very less chance you will find it in extracellular medium. If you re able to get also something its because of lysis of few cells during culture. Please find attached paper which explains in detail production of extra cellular protein in E coli.
I wonder why you'd need to have extracellular expression for these proteins? E. coli in general is not very good at secreting proteins - you'd need to pass your protein through one of the secretion systems. Sec or Tat are not enough, using these would just result with the protein ending up in the periplasm. For example, you could try fusing your protein to an autotransporter translocation domain for surface export. But if you are simply interested in getting purified protein, just express it in the E. coli cytoplasm (so without signal peptide), lyse the cells and purify. If your proteins contain disulfides, you might prefer periplasmic expression (i.e. with an E. coli signal peptide, e.g. OmpA or PelB), but then the procedure is the same (though of course you could purify just the periplasmic fraction by osmotic shock).
I would suggest that you should first confirm, whether your protein of interest is being secreted or not. Then it will be clear for you to choose method of ease for protein isolation.
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