From: Schoenfeld, Hans-J. {PRBM~Basel} <HANS-J.SCHOENFELD@Roche.COM>
  To  : ">"'Holger Strauss'" , rasmb@bbri.harvard.edu
  Date: Mon, 13 Aug 2001 12:59:16 +0200

RE: aggregates in protein samples

Dear Holger,
probably, the observed aggregates had been generated due to improper folding of a subfraction of the protein molecules. We sometimes observe such effects, when a recombinant protein was heavily overexpressed or/and when it is not authentic with respect to the primary sequences of its well soluble natural counterpart (e.g. an additional N-terminal hexahis tag).The fraction of aggregates may then contain a broad spectrum of molecular masses (starting with "dimers" and ending with visible precipitates). Often it is impossible to remove this broad distribution of artificial molecular species by simple low or high speed centrifugation. Furthermore, such preparations are often unstable, aggregates may induce further aggregation...
My recommendation:
1.) Use size exclusion chromatography as a final purification step and do not pool the first fraction(s) (ascending part) of your protein peak.
2.) Quasi-elastic light scattering (QLS; synonymously: dynamic light scattering, DLS) analysis is a most sensitive and convenient method for detection and quantification of protein aggregates. Before running AUC experiments, check your preparation by QLS and optimize your protein for monodispersity (collect only monodisperse fractions in the last steps of purification). Some further details you may get from "H.-J. Schönfeld et al., Biochemical Society Transactions, Vol. 26, pp753-758 (1998).
Consequently preserve misfolding, resp. optimize protein folding. Try to remove aggregates whenever possible during purification (QLS aided pooling, size exclusion chromatography, etc.).
If nothing else does help, a non-authentic primary sequence may exclude any chance for proper folding; then express and purify the authentic sequence (anyhow, for many other reasons this is always the best approach).
Best regards and good luck!
Hans-Joachim.
============================================
Dr. Hans-Joachim Schönfeld
F. Hoffmann-La Roche Inc.
PRBM-V, B93/5.44
CH-4070 Basel
Switzerland

Tel. (+41) 61 688 28 95
Fax. (+41) 61 688 90 60
hans-j.schoenfeld@roche.com">mailto:hans-j.schoenfeld@roche.com


	----Original Message-----
	From:	Holger Strauss [SMTP:strauss@fmp-berlin.de]
	Sent:	Friday, August 10, 2001 4:19 PM
	To:	rasmb@bbri.harvard.edu
	Subject:	aggregates in protein samples

	Dear all,

	recently we realized that we have in +- all of the protein samples we
	investigated a more or less important fraction of "unspecific"
	"aggregates" (really unspecific?), which can be seen to pile up at the
	bottom of the cell and
	diffusing back very slowly in SedEq runs, which is rather annoying since
	it interferes with the actual equilibrium we would like to look at (so we
	have to wait more time for the experiment to finish) and
	lessens the amount of data points we can reliably use for analysis.  

	We routinely dilute our samples with buffer at
	equal chemical potential for the low molecular weight components and spin
	them in the Eppendorf tube
	5 mins at 10 krpm on a usual laboratory centrifuge and do only take about
	90% of the  supernatant for actual analysis. However, this procedure
	doesn't seem to
	be sufficient in most cases, as smaller "aggregates" are more or less
	always present. 

	Any comments, suggestions, improvements for our procedures are greatly
	appreciated!

	Cheers, Holger

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	Holger Strauss 

	Forschungsinstitut fuer Molekulare Pharmakologie (FMP)
	Robert-Roessle Strasse 10

	13125 Berlin/Germany

	Tel: +49 (0)30 94793 - 223 (office)
	                     - 316 (lab)

	Fax: +49 (0)30 94793 - 169 


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	Science is spectrum analysis; art is photosynthesis.

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