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Intensification of Inclusion body Purification and Protein Refolding
Chew-Tin Lee and Anton P.J. Middelberg
Bioproducts and Bioprocessing Group,
Pembroke Street CB2 3RA, United Kingdom.
E-mail: ctl25@cam.ac.uk
The recovery of active recombinant =
proteins
from complex biological mixtures involves a series of complicated recovery
steps, each of which can compromise the purity and yield of the desired pro=
duct[1]. The elimination of unit operation=
s without
a loss of product purity is desirable to improve yield and reduce production
cost. This project aims
to develops and characte=
rises a
highly integrated reactor system for the production of refolded recombinant
proteins from inclusion bodies.
Granulocyte marcophage- colony stimulating factor (GM-CSF) expressed in E. coli<=
/span> BL21(DE3)pLysS is chosen as the=
model
protein
The conventional strategy used to r=
ecover
active protein from inclusion bodies (IBs) involves three steps: IBs isolat=
ion
and washing via homogenisation and repetitive centrifugation; solubilisatio=
n of
the IBs; and refolding of the solubilised IBs in a refolding reactor. Instead of achieving this in seve=
ral
unit operations with cumbersome multiple washing steps, we replace these
operations with a single integrated unit operation – the Integrated
Refolding and Extraction System (IRES).&n=
bsp;
IRES consists of an oscillatory flow reactor that has an integrated
hollow ceramic membrane.
Fermentation broth is added to the IRES, and chemical extraction age=
nts
are added to selectively disrupt the cell and release the insoluble IBs.byin
the IRES by operating it asby means of=
a cross-=
flow
microfiltration unit. and t<=
/del>The retent=
ate
containing the cleaned insoluble GM-CSF IBs is then solubilise=
d in
7 M guanidine hydrochloride and 50 mM DTT. The solubilised GM-CSF is subsequently perfused through =
the integrated IRES membrane into
refolding buffer that is mixed by intense oscillation.
The
three principle steps required to rea=
lise Thethe IRES are reported. These include: developed based on three major steps=
: a non-solubi=
lising chemical extraction method<=
span
class=3DmsoIns>, a cross-flow microfiltr=
ation using a ceramic membrane (Exekia, France) and refolding using the same =
membrane
as selected for microfiltration. A
non-solubilising extraction method using 0.05%=
Triton
X-100, 5 mM EDTA (in 0.=
1 M
Tris, pH 9) and intracellular T7 lysozyme has =
ins>effectively released most of the intracellular
soluble content without solubilising the GM-CSF IBs. Cross-flow microfiltration using a 0.2 <=
/span>mm cera=
mic
membrane successfully recovered the GM-CSF IBs with removal of 91 % of the
soluble contaminants and virtually no loss of IBs to the permeate. The
feed material for microfiltration was di=
luted
two-fold and treate=
d with
Benzonase. The IBs recovered from the retenta=
te of
a microfiltration run exhibited similar refolding characteristic to
those recovered by centrifugation. The viability of IRES as a refold=
ing
reactor has also been demonstrated. The integration of these <=
/ins>processes into a single unit of =
IRES demons=
trates a high level of process intensification=
for IBs purif=
ication
and protein refolding.