The method – detailed in a paper in ChemBioChem – was developed to address overexpression in bacterial systems which, according to the authors, mean processing is needed to sort correctly folded protein from aggregates – or inclusion bodies - that form.
“We report a novel method by applying finely controlled levels of shear stress to refold proteins trapped in inclusion bodies. This method might be capable of broadening the utility of bacterial overexpression, and could transform industrial and research production of proteins.”
The egg came first
The US and Australian researchers mixed a sample of boiled hens egg with a urea solution and found they could refold denatured lysozyme (HEWL) proteins by applying shear forces for 5 minutes in a piece of apparatus called a vortex flow device, or VFD.
They also showed they could reform recombinant proteins using the same approach by placing reduced-HEWL in a solution of commonly used industrial expression and purification buffers.
Other experiments with larger proteins like the catalytic domain of cAMP-dependent protein kinase A were similarly successful after the researchers modified the process to provide a closer mimic to cellular folding.
“Protein refolding by VFD requires optimization for each protein. Buffers, chaotrope additive, protein concentration, and processing time were optimized for HEWL, caveolin-ΔTM, and PKA.”
The ability to refold proteins trapped in inclusion bodies more quickly than conventional methods would have obvious cost advantages for the biopharmaceutical firms. However, the authors suggest the implications of the approach have wider implications for the drug industry.
“Most conventional processes avoid inclusion bodies by optimizing growth conditions and special cell lines at the expense of higher yields and purer protein isolated directly from bacterial cells…The continuous-flow mode of the VFD or parallel processing with multiple VFD units could allow scale-up to accommodate much larger solution volumes.
“Thus, the approach could drastically lower the time and financial costs required to refold inactive proteins on an industrial scale” they continued adding “harnessing shear forces to achieve rapid equilibration of protein folding could be expanded to a wide range of applications for research and manufacturing."
Others were less confident of the wider impact. Richard Alldread, head of innovation at CPI Biologics ,told BioPharma-Reporter.com "In my view it is an interesting development but I doubt it is going to make a commercial impact.
"The problems of expressing complex proteins in microbes such as e.coli are not just due to the problems of inclusion body refolding and even if this technology works well productivities are still likely to be lower than can be achieved with other hosts."
"There is also the issue of scaling up the technology to a suitable commercial scale which will not be simple" he continued, adding that "Therefore I would say that it has the potential to improve processes that would use inclusion body formation anyway but is not going to impact on other areas."
23 JAN 2015
“Shear-Stress-Mediated Refolding of Proteins from Aggregates and Inclusion Bodies.”
Yuan, T. Z., Ormonde, C. F. G., Kudlacek, S. T., Kunche, S., Smith, J. N., Brown, W. A., Pugliese, K. M., Olsen, T. J., Iftikhar, M., Raston, C. L. and Weiss, G. A.