Chew Tin, Lee (2003) Intensification of Inclusion body Purification and Protein Refolding. In: 6th Interlaken Conference on Advances in Production of Biologicals: From Gene to Market. , 25-30th March 2003, Interlaken, Switzerland.
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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 product. The elimination of unit operations without a loss of product purity is desirable to improve yield and reduce production cost. This project aims to develop 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 BL21(DE3)pLysS is chosen as the model protein The conventional strategy used to recover active protein from inclusion bodies (IBs) involves three steps: IBs isolation and washing via homogenisation and repetitive centrifugation; solubilisation of the IBs; and refolding of the solubilised IBs in a refolding reactor. Instead of achieving this in several unit operations with cumbersome multiple washing steps, we replace these operations with a single integrated unit operation â€“ the Integrated Refolding and Extraction System (IRES). IRES consists of an oscillatory flow reactor that has an integrated hollow ceramic membrane. Fermentation broth is added to the IRES, and chemical extraction agents are added to selectively disrupt the cell and release the insoluble IBs. Contaminants are removed by the IRES by operating it as a cross-flow microfiltration unit. The retentate containing the cleaned insoluble GM-CSF IBs is then solubilised in 7 M guanidine hydrochloride and 50 mM DTT. The solubilised GM-CSF is subsequently perfused through the IRES membrane into refolding buffer that is mixed by intense oscillation. The three principle steps required to realise the IRES are reported. These include: a non-solubilising chemical extraction method, a cross-flow microfiltration 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 Î¼m ceramic 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 diluted two-fold and treated with Benzonase. The IBs recovered from the retentate of a microfiltration run exhibited similar refolding characteristic to those recovered by centrifugation. The viability of IRES as a refolding reactor has also been demonstrated. The integration of these processes into a single unit of IRES demonstrates a high level of process intensification for IBs purification and protein refolding.
|Item Type:||Conference or Workshop Item (Speech)|
|Subjects:||T Technology > TP Chemical technology|
|Divisions:||Chemical and Natural Resources Engineering (Formerly known)|
|Deposited By:||Mohd Nazlee Faisal|
|Deposited On:||18 May 2007 10:34|
|Last Modified:||01 Jun 2010 03:07|
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