Source for plasmid expressing tagged Protein A?

Chris Larosa clarosa at biocomp.unl.edu
Sat Feb 12 06:33:28 EST 2000


Perhaps you can simply pcr it out of S. A.  designing primers based on the
published sequence, and ligate into an appropriote vector.... Sounds like
an extra week of work.

Dima Klenchin wrote:

> Greeting all,
>
> We go through tremendous quantities of Protein A linked Sepharose
> in the lab. It is expensive ($50/ml).
>
> I've heard that protein A is easily expressed in E.coli to huge amounts
> with retention of binding activity. If we could get such a vector,
> we could purify enough protein and immobilize it to the Sepharose
> very cheaply (with periodate oxydation method, it is basically a price
> of Sepharose CL-4B, protein not counting).
>
> 50 mg/liter culture --> 2 x 5 l preps --> 5 mg/ml Sepharose --> 100 ml
> sorbent which can be stored in 50% ethylene glycol at -20C essentially
> forever. Sounds like a three days worth of work.
>
> Could make a good exercize for some undergraduate student wanting
> to get his/her hands on some basic biochemical techniques.
>
> I've only seen Pharmacia vector but it needs to be modified as it does
> not have a tag or a stop codon. Pharmacia itself sells rProteinA
> Sepharose, so I figure this or a similar construct must be in someone's
> hands. An info on Protein G expression would also be helpful.
>
> Thanks for any hints,
>
>         - Dima





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