I need to purify a metalloprotein in its apo form. During heterologous expression of protein, there are chances that the protein binds to metal ions from E coli. How can I get recombinant protein from E. coli without the metal?
Dear Rashmi Bhosale
in general is it difficult to predict if a metallo protein will be produced in metallated state or not, since it depends from many factors as its metal specificity and affiinity, its expression level, the media composition and the purification protocol, since for example if you are using IMAC, it is also possible that the protein during the elution step will strip some metal from the coloumn.
in general while, if you would like to produce fully metallated protein, media supplementation with metal could be a possible solution, if the metal is not structural and your can remove it with-out induce protein unfolding, aggregation and precipitation, you can try to prepare the apo form by incubating the purified protein with a metal chelant (eg EDTA for bipositive metals as zinc, nickel, copper, BCS for monopositive metal as Cu+) and subsequentelly remove the complexed metal with a buffer exchange (by desalting of dialisys) in a metal free buffer.
In some cases if the affinity of the protein for the metal is high, reduction of pH could to protonate binding residues could be necessary to obtain the apo form.
In the past i applied the following protocol:
O/N incubation in 50 mM sodium acetate, 20 mM EDTA, pH 5.0, at 4°C, followed by protein concentration by ultrafiltration and buffer excnahge by desalting with PD10 coloumn in metal -free Tris20mM, NaCl150mM pH=8 buffer) for the production of the 2 following Zn binding protein
- apo Zmp1 (Article Identification of a Novel Zinc Metalloprotease through a Glo...
) a bacterial metallo protease- apo Human SOD1 (Article Metal-free superoxide dismutase forms soluble oligomers unde...
)N.B To be sure that you buffer is metal free, you can pass it though a metal chelating coloumn (eg chelex) or you can just add to it low EDTA (or a different chelant) concentration, if it do not affect your downtream applications.
Of course this approach work only if your protein is stable in the apo-form.
The first time, I suggest to you to perfom the metal depletion at low concentrations (max 10-20uM) so you will minimize possible aggregation propensityi in case the lost of the metal expose some hydrofobic regions.
good luck
Manuele
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