SURF222@kub.nl (Miebet) (09/19/90)
To whom it may concern:
Here follows a summary of the answers I got sofar to my question concerning
insoluble protein in a bacterial lysate after expression of a foreign protein.
Almost every answer I got dealt with inclusion bodies, that appear to be formed
in these bacteria. Nobody seems to know why they are formed. The general
solution given, is that I should dissolve the particles in GuHCl (4-6 M) and
then allow the protein to refold by either diluting the solution or by
dialising the GuHCl out. But the conditions that work for one protein,
apparently don't necessarily work for another, so a lot of work should go
into finding the appropriate refolding conditions. Two general rules are
given: 1. Keep the protein concentration relatively low (1-2mg/ml). 2. Dilute
or dialize slowly.
Two other observations that could be pertinent: 1. Don't bother at all with
denaturing and refolding if you have got enough protein anyway. 2. Maybe
lowering the temperature of the growth medium for the bugs will help preventing
the inclusion bodies to form.
Literature that was mentioned as relevant to the case:
1. Meth. in Enzym. vol. 185, 1990
2. Gene vol. 26, 109-118 (1983)
3. `Maniatis' 2nd ed., chapter 17
4. Biotechnology, vol. 6, 1214-1217 (1988)
5. Biotechnology, vol. 6, 291-294 (1988)
6. Biotechnology, vol. 7, 1141-1149 (1989)
Lastly, I want to say that I am positively surprised about the number
and friendliness of the reactions I got. In this way, I think the BIOSCI
network plays a very useful role. Thanks to everyone who took the trouble
of answering me and thanks to the guy(s) that make the network work!
Han Schilthuis
Hubrecht laboratory, Utrecht, the Nether
lands
SURF222@HTIKUB5