His-tagged (Ni-Agarose) Purification of Proteins
yuan at phoenix.princeton.edu
Thu Dec 9 11:09:22 EST 1993
Subject: Re: His-tagged (Ni-Agarose) Purification of Proteins
From: Gene Cutler, genecutl at mendel.Berkeley.EDU
Date: 8 Dec 1993 06:51:26 GMT
In article <2e3the$hl4 at agate.berkeley.edu> Gene Cutler,
genecutl at mendel.Berkeley.EDU writes:
>In article <2e3ks7$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
>the length of time of the induction often makes a difference as well as
>o.d. at the time of the induction. also i've found that the pH of the
>media as well as the pH of the sonication buffer has an effect
>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
>the effect of having the protein hanging around in the e.coli for a
>time and thus have less time to be degraded.
When we prepare our His tagged proteins in the lab we have the His tagged
vector co-transformed with a second plasmid that has the Lac I gene and
the kanamycin resistance gene. This way the His tagged protein is never
expressed until induced with IPTG. However, we have had variable results
in obtaining tagged proteins. Certain proteins are expressed very well
while others are more suspectible to degradation (despite massive
infusions of protease inhibitors) and low yields or insolubility.
However, the Lac I gene plasmid was found to be instrumental in getting
any protein expressed without killing the bacteria in many situations.
(Short digression: someone in our department had a construct that
actually lowered the O.D. of the bacteria culture within 1 hour of IPTG
induction if the bacteria did not carry the Lac I plasmid. :( )
More information about the Methods