Industrially Scaleable Process for the Purification and
Refolding of Inclusion Body Recombinant Protein
BELINDA C. CLARKE1, GARETH M. FORDE1, CHARLES HARDY2, ALEC DREW2, ROBYN O’HEHIR2
1: Department of Chemical Engineering, Melbourne, Victoria, 3800, Australia.
2: CRC for Asthma, Dept. of Immunology, Central and Eastern Clinical School, Monash University,
The Problem
The Objective
There is a large and increasing demand for the production of
recombinant protein for use in pre-clinical, clinical and commercial
applications. Traditionally, inclusion body recombinant protein is
manufactured using complicated laboratory scale unit operations.
However, the biotechnology industry is maturing and patent
protection periods are beginning to expire. This is driving a global
shift towards higher productivity, commercially viable processes.
The aim of this research is to develop, optimise and analyse a
scaleable, commercially viable process for the production of
recombinant protein from inclusion bodies. Initially, this process will
be used to produce a latex allergen (Hev b 6) for pre-clinical
studies at the CRC for Asthma. The protein needs to be of high
purity and concentration (i.e. no endotoxins), biologically active,
and in a refolded formed.
What is an inclusion body?
When E.coli is transformed to manufacture large amounts of recombinant protein, the protein sometimes forms
dense aggregates of insoluble misfolded proteins, known as inclusion bodies. The benefit from a production
aspect of inclusion bodies is that they allow high protein concentrations, protect sensitive proteins from
proteolytic (enzymatic) degradation and protect the cell from any toxic proteins. However, the challenge is to
solubilise and refold this protein into its correct ‘active’ form.
1. Fed Batch Fermentation
6. Refolding Vessel
The Process
Cell lines transformed with the
appropriate DNA supplied by the
CRC for Asthma are grown and
induced to express Hev b 6.
The protein is renatured by
decreasing the concentration of the
denaturant urea. This step will be
performed on the adsorption column
to help prevent aggregation.
2. Chemical Disruption and
Extraction
5. Expanded Bed Adsorption
Column
The cells are lysed to release the
inclusion bodies and the protein is
denatured using a chemical wash
containing urea. Spermine is also
added to reduce the negative
effects of DNA on the adsorption
column efficiency.
The protein is purified using an
automated chromatography unit
(Biorad DuoflowTM) via an affinity
agarose adsorbent (Ni charged
resin) which has specificity for the
His-tagged Hev b 6 protein.
3. Conditioning
CaCl2 is added to remove the EDTA, as EDTA decreases the
efficiency of the adsorption column.
http://web.mit.edu/king-lab/www/research/Scott/Scott-Research.html
4. Filtration
Removes precipitates and the DNA-spermine complexes. Some
concentration of the feedstock will also occur.
Preliminary Results
Protein Refolding
Chromatographic purification (2 ml packed bed) of the Hev b 6 protein has been performed as
an analytical method to facilitate optimisation studies of protein expression and to bench-mark
against the original CRC for Asthma protocol. These results will determine optimal operating
parameters (e.g. expression time, induction cell density, inducer concentration) for the scaleable
protein production process.
The most significant challenge of this
process is the refolding step as;
▪ Small amounts of contaminants can
decrease the yield of refolded protein
(for example, contaminants can cause
aggregation and proteases cause
degradation of the protein).
▪ The protein must be in an
environment
that
favours
the
formation of the correctly folded
proteins over the misfolded. This may
require chaperones, metal ions etc.
▪ Refolding conditions vary with
proteins.
Chromatograph for Purification of Hev b 6-His Tagged Protein
Indication of protein
concentration
leaving column
Fractions collected and analysed using Bradford
Assay and SDS-Page to identify the products
Bound protein eluted
(total of approximately 3.8 mg
protein from Bradford Assay)
Acknowledgement: