His-tagged (Ni-Agarose) Purification of Proteins
genecutl at mendel.Berkeley.EDU
Wed Dec 8 01:51:26 EST 1993
In article <2e3ks7$iaf at news.bu.edu>, Richard Near <rocket1 at bu.edu> wrote:
>I can sympathize with this problem. I'm trying to express a
>peptide in the Novagen vector pET19b which includes a His-tag.
>I'm also having low yields. The advice I've gotten from Novagen
>and fellow sufferers is 1: Make sure you're not losing the plasmid
>by working from a freshly subcloned colony and using carbinicilin
>and not overgrowing the bugs, 2: Try growing your bugs at different
>temp, 3: I presume you're assaying the inclusion bodies as well as
>soluble proteins, as this is often where the protein accumulates, and
>4: Try adding protease inhibitors when lysing the bacteria (also
>assay several time points after induction).
it seems like this is a protein expression problem and not a problem with
the his-tag. one thing that often makes a difference is the induction
condition. some proteins express better if less iptg is added, others more.
the length of time of the induction often makes a difference as well as the
o.d. at the time of the induction. also i've found that the pH of the growth
media as well as the pH of the sonication buffer has an effect (especially
on the solubility of the protein).
if you have a lot of breakdown product in the crude e. coli extract,
i would recomend inducing for a shorter time, perhaps at a higher o.d. to
counteract the lower level of expression that would result. this would have
the effect of having the protein hanging around in the e.coli for a shorter
time and thus have less time to be degraded.
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