Responses for solubilization of membrane proteins !

G. Jawahar Swaminathan jawahar at nii.ernet.in
Thu Apr 14 02:18:42 EST 1994


Dear Netters,

I had posted an enquiry about the way to go about starting work on
solubilization of membrane proteins ( buffer conditions, pH, detergent etc).
Thank you for your responses on this subject; with the ideas received I think
we have a starting base to standardize our own techniques. I am posting the 
replies I received from various people on this subject below.
Thanx once again !!
I shall post summary of responses as they accumlate to restrict the size of the
postings !! :-)
jawahar at nii.ernet.in
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From:	gchacko at magnus.acs.ohio-state.edu

In article <9404051542.AA24196 at ern.doe.ernet.in> you write:

>This is a request from a friend of mine who does not have access to the email
>facility. He is starting to work on the purification of a membrane bound
>protein from macrophage cell line. Does anyone have any idea as to how to go
>about the process of solubilize the membrane, i.e., detergents, buffer, pH etc
>for such a protein. The protein has to studied later by transferring it into a
>liposome enviornment. 

1% Triton-X 100 in 100mM PBS PH 7.4 and 10mM EDTA is a very
conservative way to start. We use 2mM PMSF, 20ug/ml leupeptin and
10ug/ml aprotinin as well to keep protease activity down. This can get
expensive for large scale preps. If you're looking at phosphorylated
proteins you can add 1mM sodium orthovanadate. You should be able to
find all this in the literature quite easily.

GC
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From:	jthom at WUMS.WUstl.EDU
We saw your message on bionet.molbio.methds-reagnts about solubilizing a
membrane-bound protein from macrophage.  The following method was used to
solubilize 3beta-hydroxysteroid dehydrogenase/isomerase from human
placental microsomes (James L. Thomas et al, J Steroid Biochem 31:785-793,
1988).

Placental microsomes (10 mg protein/ml) in 0.1 M potassium phosphate
buffer, pH 7.5, 20% glycerol, 0.1 mM EDTA were stirred with 0.6% sodium
cholate on ice for 30 minutes.  The mixture was then centifuged at 120,000
x g for 1 hour.  The supernatant contained 50% of total 3beta-HSD/isomerase
activity, and the solubilized enzyme was purified to homogeneity in two
steps.  We obtained a higher per cent solubilization with the nonionic
detergent, Brij 58, but the enzyme was bound to detergent micelles and
could not be purified further.

We also prepared liposome-bound enzyme using phospholipids extracted from
asolectin.  The key to success was removal of detergent using a column of
Bio-Beads SM-2 (Bio-Rad), then eluting the enzyme out the beads with a
liposome mixture. Liposome references: JP Wehrle & RM Pollack, Steroids 47:115,
1986; DS Bracy et al, Biochim Biophys Acta 899;51-58, 1987; J Sunamoto et
al, Ann NY Acad Sci 613:116-127, 1990.
===============================================================================
From:	maraman at ksu.ksu.edu  
 Hi, I read your mail in the internet.  To deal with the membrane 
proteins, first you have to know whether it is an integral or
peripheral membrane protein.  If it is an integral membrane 
protein , then you have to get those membranes purified first, like   
plasma membranes or organelle membranes.  Then try a panel of 
detergents which have a very high c.m.c. like Octyl glucoside,Decyl
maltoside, Dodecyl maltoside, CHAPS, or ionic detergents like 
sodium cholate, deoxy cholate.  Some use even digitonin. Try from 
1x c.m.c to 10x c.m.c for each detergent you choose, extract your 
membranes, take the supernatant & assay for some kind of activity
to know whether your protein is alive or not. Look for maximal 
yield with optimal activity of your protein. Fix these parameters
& then put it in model membranes & study the orientation of your
protein. Theoretically it is 50% inside out & 50% right side out
vesicles. You can try triton x-100 also but you need SM-2 biobeads
to remove them. But if you do any flourescence spec. on the 
reconstituted preparation it is useless. Well, good luck, if you 
need more help e-mail me at maraman at matt.ksu.ksu.edu.Bye now.
============================================================================
From:	krasel at alf.biochem.mpg.de
To solubilize beta2-adrenergic receptor from Sf9 membranes, we do the
following:

- prepare membranes
- resuspend 40 ml membranes (8 mg protein/ml) in 100 ml 20 mM Tris pH 7.4
  50 mM NaCl x% Laurylsucrose (final concentration of Laurylsucrose has to
  be 0.4%)
- stir slowly in cold room for 45 min
- centrifuge at 18000 rpm for 30 min in SS34
- supernatant contains the solubilized receptor

The receptor is then purified by alprenolol affinity chromatography and
reconstituted into liposomes.

Your friend will probably have to try different detergents.

Hope that helps,
--Cornelius.




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