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.

Active membrane transport and receptor proteins from bacteria

A general strategy for the expression of bacterial membrane transport and receptor genes in Escherichia coli is described. Expression is amplified so that the encoded proteins comprise 5-35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGSH6 or a Strep tag at the C-terminus. These enable purification in mg quantities for crystallization and NMR studies. Examples of one nutrient uptake and one multidrug extrusion protein from Helicobacter pylori are described. This strategy is successful for membrane proteins from H. pylori, E. coli, Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Microbacterium liquefaciens, Brucella abortus, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides, Streptomyces coelicolor and Rhodobacter sphaeroides. ©2005 Biochemical Society.

Fish farm effluent control and the development of an expert system

In recent years, freshwater fish farmers have come under increasing pressure from the Water Authorities to control the quality of their farm effluents. This project aimed to investigate methods of treating aquacultural effluent in an efficient and cost-effective manner, and to incorporate the knowledge gained into an Expert System which could then be used in an advice service to farmers. From the results of this research it was established that sedimentation and the use of low pollution diets are the only cost effective methods of controlling the quality of fish farm effluents. Settlement has been extensively investigated and it was found that the removal of suspended solids in a settlement pond is only likely to be effective if the inlet solids concentration is in excess of 8 mg/litre. The probability of good settlement can be enhanced by keeping the ratio of length/retention time (a form of mean fluid velocity) below 4.0 metres/minute. The removal of BOD requires inlet solids concentrations in excess of 20 mg/litre to be effective, and this is seldom attained on commercial fish farms. Settlement, generally, does not remove appreciable quantities of ammonia from effluents, but algae can absorb ammonia by nutrient uptake under certain conditions. The use of low pollution, high performance diets gives pollutant yields which are low when compared with published figures obtained by many previous workers. Two Expert Systems were constructed, both of which diagnose possible causes of poor effluent quality on fish farms and suggest solutions. The first system uses knowledge gained from a literature review and the second employs the knowledge obtained from this project's experimental work. Consent details for over 100 fish farms were obtained from the public registers kept by the Water Authorities. Large variations in policy from one Authority to the next were found. These data have been compiled in a computer file for ease of comparison.