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Purification of metallothioneins? - (Sep/19/2014 )

hi to all,

i have expressed in e.coli a 6x his-tagged protein having 2 cysteine rich heavy metal binding domains. i have performed an imac purification and by examining the various fractions with an electrophoretic gel, there is a mess: it seems that the protein is aggregated.

the elution buffer was composed by Na2HPO4 20mM, NaCl 250mM, ZnSO4 0.05mM, EDTA 50mM and imidazole 10mM, pH=8 and in each fraction 2mM DTT was added. 

the electrophoresed samples consisted of the eluted fraction containing the supposed protein and of laemmli 1x with SDS, glycerol, DTT and so on.

how could I manage this issue?

do you think that an enzymatic digestion with the xa factor in ordes to remove the his tag would help?

thank you,

Antonio

-Antonio Sillo Rosato-

Why do you have so much EDTA in your buffer?...that much will chelate off the nickel so your imac will never work. If your buffer really has 50mM EDTA, your protein is flowing right through the column.

 

Also, aggregation in your sds-page is not necessarily indicative of aggregation in solution. It likely means either your protein aggregates in the presence of SDS or your gel ran funny. Typically with soluble proteins, SDS will monomerize your protein rather than cause aggregates.

 

I would redo the his-tag purification without the EDTA, or with EDTA < 1mM. Also consider using mercaptoethanol in all your buffers (10mM is safe for ni-nta) to ensure your cystein rich protein isn't forming disulfide-linked oligomers.

 

Removal of your his-tag by digestion will likely not help at this point. I think your problems lie "upstream" (experimentally that is) of that step. Consider optimizing your buffer with something that could stabilize it (detergent , glycerol, different pH, more/less salt, etc). Expression at low temps can (though not always) improve protein behavior. Also consider cleavable stabilization/solubilization tags (SUMO, GFP, MBP, GST, etc).

 

Hope that helps some.

-labtastic-