Source for plasmid expressing tagged Protein A?

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Mon Feb 14 21:26:13 EST 2000


In article <38A54508.38187EEC at biocomp.unl.edu>, Chris Larosa <clarosa at biocomp.unl.edu> wrote:
>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.

I don't want/need it _that_ much to make it my side project (which 
might actually take more than one week; crap happens), particularly
when it is _known_ such vectors exist (the fist time I heard about fusion
proteins in E.coli was in ~1986 and that was a Protein A fusion). If I were
a hobbist at home, I'd say this is a very worthwhile project :-)

        - Dima

>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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