Developing a scalable model of recombinant protein yield from Pichia pastoris:the influence of culture conditions, biomass and induction regime

Background The optimisation and scale-up of process conditions leading to high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences. Typical experiments rely on varying selected parameters through repeated rounds of trial-and-error optimisation. To rationalise this, several groups have recently adopted the 'design of experiments' (DoE) approach frequently used in industry. Studies have focused on parameters such as medium composition, nutrient feed rates and induction of expression in shake flasks or bioreactors, as well as oxygen transfer rates in micro-well plates. In this study we wanted to generate a predictive model that described small-scale screens and to test its scalability to bioreactors. Results Here we demonstrate how the use of a DoE approach in a multi-well mini-bioreactor permitted the rapid establishment of high yielding production phase conditions that could be transferred to a 7 L bioreactor. Using green fluorescent protein secreted from Pichia pastoris, we derived a predictive model of protein yield as a function of the three most commonly-varied process parameters: temperature, pH and the percentage of dissolved oxygen in the culture medium. Importantly, when yield was normalised to culture volume and density, the model was scalable from mL to L working volumes. By increasing pre-induction biomass accumulation, model-predicted yields were further improved. Yield improvement was most significant, however, on varying the fed-batch induction regime to minimise methanol accumulation so that the productivity of the culture increased throughout the whole induction period. These findings suggest the importance of matching the rate of protein production with the host metabolism. Conclusion We demonstrate how a rational, stepwise approach to recombinant protein production screens can reduce process development time.

Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

Background The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in high-yielding protein production hosts. Results We show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets. Online flow microcalorimetry demonstrated that there had been a substantial metabolic change to cells cultured under high-yielding conditions, and in particular that high yielding cells were more metabolically efficient. Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis. Conclusion This work demonstrates the power of a rational approach to recombinant protein production by using the results of transcriptome analysis to engineer improved strains, thereby revealing the underlying biological events involved.

An exploratory study to determine applicability of nano-hardness and micro-compression measurements for yield stress estimation

The superior properties of ferritic/martensitic steels in a radiation environment (low swelling, low activation under irradiation and good corrosion resistance) make them good candidates for structural parts in future reactors and spallation sources. While it cannot substitute for true reactor experiments, irradiation by charged particles from accelerators can reduce the number of reactor experiments and support fundamental research for a better understanding of radiation effects in materials. Based on the nature of low energy accelerator experiments, only a small volume of material can be uniformly irradiated. Micro and nanoscale post irradiation tests thus have to be performed. We show here that nanoindentation and micro-compression testing on T91 and HT-9 stainless steel before and after ion irradiation are useful methods to evaluate the radiation induced hardening.

Acoustic emission and yield in sand

An experimental investigation into the Acoustic Emission (AE) response of sand has been undertaken, and the use of AE as a method of yield point identification has been assessed. Dense, saturated samples of sand were tested in conventional triaxial apparatus. The measurements of stresses and strains were carried out according to current research practice. The AE monitoring system was integrated with the soil mechanics equipment in such a way that sample disturbance was minimised. During monotonically loaded, constant cell pressure tests the total number of events recorded was found to increase at an increasing rate in a manner which may be approximated by a power law. The AE response of the sand was found to be both stress level and stress path dependent. Undrained constant cell pressure tests showed that, unlike drained tests, the AE event rate increased at an increasing rate; this was shown to correlate with the mean effective stress variation. The stress path dependence was most noticeable in extension tests, where the number of events recorded was an order of magnitude less than that recorded in comparable compression tests. This stress path dependence was shown to be due to the differences in the work done by the external stresses. In constant cell pressure tests containing unload/reload cycles it was found that yield could be identified from a discontinuity in the event rate/time curve which occurred during reloading. Further tests involving complex stress paths showed that AE was a useful method of yield point identification. Some tests involving large stress reversals were carried out, and AE identified the inverse yield points more distinctly than conventional methods of yield point identification.

A study of the micro-mechanics of granular media

The development of more realistic constitutive models for granular media, such as sand, requires ingredients which take into account the internal micro-mechanical response to deformation. Unfortunately, at present, very little is known about these mechanisms and therefore it is instructive to find out more about the internal nature of granular samples by conducting suitable tests. In contrast to physical testing the method of investigation used in this study employs the Distinct Element Method. This is a computer based, iterative, time-dependent technique that allows the deformation of granular assemblies to be numerically simulated. By making assumptions regarding contact stiffnesses each individual contact force can be measured and by resolution particle centroid forces can be calculated. Then by dividing particle forces by their respective mass, particle centroid velocities and displacements are obtained by numerical integration. The Distinct Element Method is incorporated into a computer program 'Ball'. This program is effectively a numerical apparatus which forms a logical housing for this method and allows data input and output, and also provides testing control. By using this numerical apparatus tests have been carried out on disc assemblies and many new interesting observations regarding the micromechanical behaviour are revealed. In order to relate the observed microscopic mechanisms of deformation to the flow of the granular system two separate approaches have been used. Firstly a constitutive model has been developed which describes the yield function, flow rule and translation rule for regular assemblies of spheres and discs when subjected to coaxial deformation. Secondly statistical analyses have been carried out using data which was extracted from the simulation tests. These analyses define and quantify granular structure and then show how the force and velocity distributions use the structure to produce the corresponding stress and strain-rate tensors.