Source for plasmid expressing tagged Protein A?

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Fri Feb 11 17:27:16 EST 2000


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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