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Coexpression of Chaperones/Foldases make Properly folded recombinant proteins

Coexpression of Chaperones/Foldases make Properly folded recombinant proteins - Recombinant Protein Forum

Coexpression of Chaperones/Foldases make Properly folded recombinant proteins - Recombinant Protein Forum. Discuss purification, expression, and isolation of recombinant and cloned proteins here.


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Old 10-27-2009, 08:08 PM
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Post Coexpression of Chaperones/Foldases make Properly folded recombinant proteins



If you ever wanted "chaperones" to properly fold you recombinant proteins, check out this article:
Expression of correctly folded proteins in Escherichia coil
George Georgiou* and Pascal Valaxt

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Abstract: Many heterologous polypeptides fail to fold into their native
state when expressed in Escherichia co~i; instead, they
are either degraded by the cellular proteolytic machinery
or accumulate in insoluble form, typically as inclusion
bodies. Misfolding is a particularly vexing problem in the
expression of mammalian proteins, especially those that are
composed of multiple subunits, have several disulfide bonds,
or contain prosthetic groups. Fortunately, bacteria exhibit a
remarkable physiological plasticity that can be successfully
exploited to improve protein folding. Significant yields of
active heterologous proteins have been obtained through
strategies that include the co-expression of homologous or
heterologous folding accessory proteins, the optimization of
growth conditions, and the use of fusion proteins. A flood
of recent reports documenting the successful production of
complex eukaryotic proteins in active form have demonstrated
that bacteria can provide the proper environment for the
folding of the vast majority of recombinant polypeptides.
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Old 10-27-2009, 08:38 PM
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Post Re: Coexpression of Chaperones/Foldases make Properly folded recombinant proteins

Another similar article:
Folding and assembly of oligomeric proteins in
Escherichia coil
Carolyn M. Teschke and Jonathan King


Also mentions the optimization of temperature and ionic strength of media for greater recovery. See excerpts.

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Excerpt 1: The cytoplasm of E. coil
David Goodsell [23] calculated the contents of a 100 nm
cube of E. coli cytoplasm, one of 600 in an E. coli
cell. The cube contained 30 ribosomes, 2-3 mRNA
molecules and 6 RNA polymerases, 100 protein translation
factors, 330 other protein molecules, 30 amino
acyl-tRNA synthetases, 340 tRNA molecules, 30000
small molecules such as precursors and cofactors, and
50 000 small ions. The turgor pressure of the cell causes
the internal pressure to be 3 atm. The volume of the
cytoplasm, the amount of H20 contained in that volume
and the concentration of protein in the cytoplasm,
are all dependent on the osmolarity of the medium in
which the cells are grown [24].

The bacterium responds to changes in the osmolarity
of the medium by altering its internal K + and glutamate
ion concentration, as well as adjusting the volume of
its cytoplasm. The volume of the cytoplasm decreases
twofold with an increase in medium osmolarity from
0.1 OsM to 1.02 OsM, or 0M NaC1 to 0.5M NaCl, in the
medium of Cayley et al. [24]. The decrease in the volume
of the cytoplasm is accompanied by an increase
in the intracellular concentration of osmolytes, mostly
the K + ion concentration. The protein concentration increases
from 200 mg ml- 1 to 340 mg ml- 1 and the RNA
concentration from 75 mg ml- 1 to 120 mg ml- 1 over the
range of osmolarities investigated, leading to a 1.6-fold
increase in macromolecule concentration. Clearly the
local environment of newly synthesized proteins is
highly concentrated. These protein concentrations in
vitro cause excluded volume nonideality and can have
major effects on macromolecular processes [24].

Excerpt 2: Environmental alterations to increase the recovery of oligomers
Two variables under experimental control in vivo are
the temperature and ionic strength of the medium.
When formation of inclusion bodies is due to thermolability
of folding intermediates in the bacterial cytoplasm,
lowering the temperature of cell growth is an
obvious path to increasing the yield of correctly folded
protein [6,14]. Additional methods for improving yield,
such as decreasing the growth rate, have recently been
reviewed by Schein [6].

The internal ionic strength can be altered by increasing
the osmolarity of the medium. The increased concentration
of cellular protein stabilizes the protein subunits,
much as glycerol or sucrose do in vitro, by
changing the activity of the I-I20. In fact, osmoremedial
mutations, where altered proteins are nonfunctional at
low osmolarities but become more functional if the osmolarity
of the medium is increased, are common [25].
Many temperature-sensitive mutations are also osmoremedial.
This is probably not due to changes in the
internal ions in the cytoplasm but may be caused by
effects on water activity [24].
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