CORRECTION: "Eukaryotes" changed to "Prokaryotes". Vernon
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Jeff-
Thanks for your suggestion about centrifugation. However, one thing I didn't say was that our protein is expressed as small inclusion bodies. We separate the inclusion bodies (and ribosomes) from the supernatant by ultrafiltration, and then disrupt the inclusion bodies (and presumably the ribosomes) with a denaturant.
As a point of interest, for development of purification processes in industry, the developers often have to make sure their procedures are scalable, i.e. what will work at small scale should work at large scale. Unfortunately, high-speed centrifugation is not one of those!
Actually, I'd appreciate seeing some references about isolation (lab scale!) of whole ribosomes from prokaryotes. The ribosome literature is large, and this isn't my area of expertise.
Vernon
Vernon A. Shoup
Regeneron Pharmaceuticals, QC Dept.
81 Columbia Tnpk.
Rensselaer, NY 12144
(518)488-6012
vernon.shoup@regpha.com
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Date: 8/12/98 9:06 AM
To: VERNON SHOUP
From: Jeffrey A Kowalak
>We are producing an E.coli-derived recombinant protein drug candidate that
>has a pI of about 10. Because of this high pI (positive charge under
>physiological conditions), ribosomal proteins tend to copurify with it.
>Eventually a host cell protein assay that will detect these proteins will
>be needed. We plan to send some empty vector material part way through
>the purification process to isolate the proteins, but in the interim...
>
>We're interested in obtaining either some purified E.coli ribosomes, or
>more preferably the set of proteins isolated from the ribosomes.
>Analytical quantities would be OK for now. Does anyone know where I can
>obtain this material?
>To push this a bit more, we'd also love to get hold of some polyclonal
>antibodies that would recognize a range of E.coli ribosomal proteins.
>Does anybody have any suggestions? If these materials are available
>privately, we'd be glad to arrange payment.
>
Hi Vernon,
Have you considered simply removing the ribosomes by centrifugation
(1.0 M sucrose, properly buffered, should do nicely). The 70S ribosomes
will pellet, then start purifying your protein from the supernatant. I can
provide refs if you need them.
Regards,
Jeff Kowalak
Jeffrey A Kowalak, Ph.D.
jkowalak@codon.nih.gov
Section on Metabolic Analysis and Mass Spectrometry
National Institute of Child Health and Human Development
Building 10 Room 9D52
Bethesda MD 20892-1580
telephone: 301-594-3678
facsimile: 301-480-5460