Source for plasmid expressing tagged Protein A?

a.krimer at biosidus.com.ar a.krimer at biosidus.com.ar
Mon Feb 14 08:42:56 EST 2000


Dear Dima:
Why you don't try using the Staphylococcus aureus strain producing protein A
(the name is Cowan strain, I believe).
Several years ago I ve purified from this strain and on this way you avoid
cloning steps.
Hope this helps

Alejandro krimer





klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) on 02/11/2000
07:27:16 PM

To:   methods at hgmp.mrc.ac.uk, proteins at hgmp.mrc.ac.uk
cc:    (bcc: Alejandro Krimer/DESARROLLO/BIOSIDUS/GRUPOSIDUS)
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Subject:  Source for plasmid expressing tagged Protein A?



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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