Hi there,

In most cases zinc metalloprotease is able to trap zinc during induction/expression in rich medium and if interaction is strong enough, zinc remains bound to the protein during the purification process without the need of any additive. If working in minimal media then zinc has to be added during the culture. If you want to be sure zinc binding site remains occupied during purification supplement any of the buffers with zinc salt. Tens of µM should be enough.

Dear Madeleine

i'm agree with Dominique about zinc addiction in the culture and the concentration of zinc to add .

However in my experience in some cases the presence of the his tag and the purification with an imac coloumn where metals different from zinc ( as nickel) are immobilized on the coloumn can produce protein partially metallated or binding the wrong metal.

this for example happened to me with clostridium zmp1 ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841139/) where to obtain the active zinc form, incubation with edta at low ph was required to remove the nickel and replace it with zinc.

so if you have in your hand an activity assay try to perform it with the puried protein supplemented with zinc and not but in case this not work, try to produce app protein and re-metallated it adding zinc in different ratio (eg 1:1, 1:2 and 1:10 in terms of milar concentration)

good luck

Manuele

Madeleine,

During purification, you typically try to avoid conditions that could allow endogenous proteases to degrade your overexpressed product (by using EDTA and protease inhibitors in the lysis buffers and first few purification steps). Thus, your purified protease prep would contain little divalent cation, including Zn++.

It is likely that you will get enzymatic activity by diluting your purified protease in reaction buffer containing ZnCl2 (1, 2, 4 mM, or maybe even up to 10 mM) and your substrate. The enzyme is diluted considerably in buffer (allowing divalent cations to start dissociating from the active site), and the mM levels of Zn++ will likely engage the metal-binding site(s) of the active site resulting in active enzyme.

Best,

Mark