MIME-Version: 1.0 Content-Location: file:///C:/237451CB/InterLK.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="macintosh" Intensification of inclusion body purification and protein refolding=

Intensification of Inclusion body Purification and Protein Refolding


Chew-Tin Lee and Anton P.J. Middelberg

Bioproducts and Bioprocessing Group,

Department of Chemical Engineering, University of Cambridge

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.  Contaminants are removed bybyin 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 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.