Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort

A renaissance in cell-free protein synthesis(CFPS)is underway,enabled by the acceleration and adoption of synthetic biology methods.CFPS has emerged as a powerful platform technology for synthetic gene network design,biosensing and on-demand biomanufacturing.Whilst primarily of bacterial origin,cell-free extracts derived from a variety of host organisms have been explored,aiming to capitalise on cellular diversity and the advantageous properties associated with those organisms.However,cell-free extracts produced from eukaryotes are often overlooked due to their relatively low yields,despite the potential for improved protein folding and posttranslational modifications.Here we describe further development of a Pichia pastoris cell-free platform,a widely used expression host in both academia and the biopharmaceutical industry.Using a minimised Design of Experiments(DOE)approach,we were able to increase the productivity of the system by improving the composition of the complex reaction mixture.This was achieved in a minimal number of experimental runs,within the constraints of the design and without the need for liquid-handling robots.In doing so,we were able to estimate the main effects impacting productivity in the system and increased the protein synthesis of firefly luciferase and the biopharmaceutical HSA by 4.8-fold and 3.5-fold,respectively.This study highlights the P.pastoris-based cell-free system as a highly productive eukaryotic platform and displays the value of minimised DOE designs.

Protein construct optimization:data sharing strategy

The British Medical Journal devoted its first issue of 2012 to the problem of unreported clinical trial data,highlighting the serious impact that incomplete information can have on world health(for an editorial summary see Lehman and Loder,2012).Adopted innovations in clinical intervention,inappro-priately based on only a subset of trial data,can,at best,prove uneconomical and,at worst,lead to large-scale patient death.While its consequences may be most severe in medicine this problem applies to many scientific disciplines and we are now seeing a move towards greater transparency in data declaration.

Investigating the consequences of asymmetric endoplasmic reticulum inheritance in Saccharomyces cerevisiae under stress using a combination of single cell measurements and mathematical modelling

Adaptation allows organisms to maintain a constant internal environment,which is optimised for growth.The unfolded protein response(UPR)is an example of a feedback loop that maintains endoplasmic reticulum(ER)homeostasis,and is characteristic of how adaptation is often mediated by transcriptional networks.The more recent discovery of asymmetric division in maintaining ER homeostasis,however,is an example of how alternative non-transcriptional pathways can exist,but are overlooked by gold standard transcriptomic or proteomic population-based assays.In this study,we have used a combination of fluorescent reporters,flow cytometry and mathematical modelling to explore the relative roles of asymmetric cell division and the UPR in maintaining ER homeostasis.Under low ER stress,asymmetric division leaves daughter cells with an ER deficiency,necessitating activation of the UPR and prolonged cell cycle during which they can recover ER functionality before growth.Mathematical analysis of and simulation results from our mathematical model reinforce the experimental observations that low ER stress primarily impacts the growth rate of the daughter cells.These results demonstrate the interplay between homeostatic pathways and the importance of exploring sub-population dynamics to understand population adaptation to quantitatively different stresses.