Characterisation of recombinant protein solubilisation and refolding.
Summer Studentship Report 2024.
By Marios Zare, Mark Carlile- University of Sunderland.
Since the early 1980s, recombinant protein production has been an established
method for the generation of industrial enzymes and protein therapeutics. However, the
complexity of some proteins and the associated processing costs has meant that some products
have not been fully exploited. The E.coli expression system is a cheap alternative to the costly
mammalian expression platforms, but is unable to generate large amounts of complex proteins.
The expression of proteins in an insoluble format (inclusion bodies) followed by solubilisation
and refolding is one way to express more complex proteins to a higher titre.
In this short project an E.coli expression system was generated for the generation of
ribosomal protein L1, which was used as a model protein for solubilisation and refolding
studies. Cultures ranging from 10-200 mL were generated and progressed through to filtered
refold material for loading onto a capture chromatography column. POROS RP-HPLC was
used to profile the expression level and purity of the RPL1 protein throughout the purification
process.
A protocol was generated for the processing of RPL1 inclusion bodies
was achieved. An expression titre screen shoed a variability in the expression level of RPL1
and the biomass aligned with each expression clone. The highest producing strain was used to
scale-up expressions and inclusion body preparations for subsequent solubilisation and
refolding experiments. The protein concentration at the solubilisation step was varied between
10-20 mg/mL. The solubilisation and refolding of this material shown no significant difference
in product quality and a small positive difference in efficiency as the amount of protein in the
solubilisation increases.
This work has successfully generated RPL1 material that progressed through a
capture chromatography step from inclusion bodies expressed in E.coli. The work allowed a
protocol to be generated for each processing step unto an including the capture
chromatography. Preliminary data has been generated for subsequent studies on this and other
recombinant proteins.
ChromSoc/BMSS Summer Studentship program information.
