On Fri, 11 Feb 2000, 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.
Dima:
Modifying the Pharmacia vector could end up being quite a bit of work.
Why not use the formalin-fixed Staph aureus cowan strain itself? By
comparison it is far cheaper than buying already conjugated protein A
sepharose or purified protein A which you can couple yourself to CNBr
activated sepaharose beads (even more so if you have to activate the beads
yourself, which is rather toxic!).
It's been a while, but if memory serves we got the fixed bugs from Gibco.
Also, you can get the S. aureus cowan strain from ATCC, but obviously care
must be taken so that one doesn't start a local "staff" infection of the
staff! Basically one grows the bugs to a certain concentration and then
proceed to wash and incubate them in a variety of "para-formaldehyde"
solutions until they are all fixed.
We used to do our immuno-precips with the store-bought bugs and had to
wash the 100 mls of bugs in a SDS/BME solution to "prep" them for IPs to
reduce non-specific background. I never did try the fixed bugs for Ig
purification thought -- I always reused protein A sepharose for that.
Hope this helps,
David
==========================
David L. Haviland, PhD
Assistant Professor, Immunology
University of Texas, HSC - Houston
Institute of Molecular Medicine
2121 W. Holcombe Blvd.
Houston, TX 77030
http://www.uth.tmc.edu/~dhavilan
713.500.2413-Voice
713.500.2424-FAX
==========================
---
