Soluble Protein with pEt-Sorbitol/Betaine

Bradley Turner bturner at MED-ITVAX1.BU.EDU
Fri Apr 5 18:07:58 EST 1996


Hello Tina Hallis,

The following system was mentioned on the "net" a while back.  I don't
have the original post, but basically it involves growing your
expressing bacteria in LB media plus appropriate antibiotics, IPTG
and 1M Sorbitol and 2.5mM Betaine.  According to the references below
the E coli accumulate high intracellular concentrations of betaine
in response to the high extracellular sorbitol concentrations.  This
high concentration of intracellular betaine is able to stabilize
expressed proteins causing a shift from insoluble/inactive forms (i.e
inclusion bodies) to soluble/active forms (in the cytosol).  The
first reference describes a >400 fold increase in expressed enzyme
activity using this system.

Caveat: I have only started to use this system and have not done any
comparisons yet, but I have noticed that my bacteia (BL21-DE3 containing
pRSET a pET derivative) grow considerably slower in this system.

Here are the references:

"A novel strategy for production of a highly expressed recombinant
protein in an active form."
John R. Blackwell and Roger Horgan
FEBS Letters 295(1,2,3):10-12, 1991 Dec

"Recent developments in heterologous protein production in Escherichia
coli"
Robert C. Hockney
Trends in Biotechnology 12:456-63, 1994 Nov

" The stabilization of proteins by osmolytes"
T. Arakawa and S.N. Timasheff
Biophysical Journal 47:411-414, 1985 Mar

"Adaptation of Escherichia coli to high osmolarity environments:
Osmoregulation of the high-affinity glycine betaine transport system
ProU"

Jan M. Lucht and Erhard Bremer
FEMS Microbiology Reviews 14:3-20, 1994

"Osmotic regulation of gene expression: ionic strength as an
intracellular signal?"
Christopher F. Higgins, John Cairney, Douglas A. Stirling,
Lesley Sutherland and Ian R. Booth
Trends in Biochemical Sciences 12:339-345, 1987 Sep

I hope this helps,


Brad Turner

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		    Bradley Turner
	         Beth Israel Hospital

Harvard Medical School		617-667-1215 phone
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Boston, MA 02215 USA		bturner at med-itvax1.bu.edu
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