- Oral Presentation
- Open Access
Impact of high throughput technology on recombinant protein production
© Jones et al; licensee BioMed Central Ltd. 2006
- Published: 10 October 2006
- Recombinant Protein
- Cold Storage
- Host Strain
- Recombinant Protein Production
There are many challenges still to be overcome in the efficient production of recombinant protein from both eukaryotic and prokaryotic organisms. Often the problems faced are protein specific, and can result in unacceptable time delays between target screening and structural elucidation. A requirement therefore exists for rapid, parallel approaches that can identify suitable clones under a preliminary set of fermentation conditions in days rather than months.
Piccolo® is a fully automated, high throughput system, specifically developed for the rapid optimisation of protein production from both insect and microbial cells. The system is capable of expressing a maximum of 1152 cultures, at volumes between 10 ml and 100 mls, followed by protein purification using affinity chromatography. A total of four different culture media, two antibiotics and two inducers can be used, in conjunction with three post induction temperatures. The optical density of cultures is measured robotically to monitor growth, and the growing cultures can be both aerated and agitated continuously to prevent anaerobic growth. Following expression, the cells are centrifuged, lysed and the lysate bound to affinity resin. The resin is then washed, eluted and the partially purified protein stored at 6°C ready for analysis.
Results are presented that show the increased efficiency of combining the automated primary screening of Piccolo® with on-line GFP sensors to optimise the subsequent fermentation processes. The use of an on-line sensor to measure GFP-tagged protein expression in fermenters has been described previously by Jones et al. . In the pharmaceutical industry, reducing the time taken for screening recombinant protein is becoming more important and this automated approach will undoubtedly have an important role.
Early results from experiments completed using Piccolo® are encouraging. To illustrate the potential of the technology, an experiment to investigate different host strains of E. coli was undertaken. The expression conditions were induction at an optical density of 1.0 with 0.5 mM IPTG followed by incubation for 7 h at 30°C and for 3 h at 37°C. Six proteins all with an N-terminal 6-histidine tag were evaluated in three different host strains. The experiment used a total of 720 individual cultures, with samples in quadruplicate. Cultures were grown in special custom built culture vessel blocks (CVBs) containing 24 wells per block fitted with an aeration assembly that allowed both addition of gases and agitation. On-line growth measurements using an optical sensor were also possible. The culture blocks were incubated before induction at 37°C, moved to a post-induction incubator at the desired induction statistic for 30 minutes. After 30 minutes the CVB was removed from the incubator, IPTG was added and the CVB returned for incubation for 3 h at 37°C or 7 h at 30°C. The CVBs were removed, decoupled from their aeration assemblies and moved to cold storage at 6°C until ready for purification.
The potential of a high throughput automated system for industry to screen a large number of conditions is demonstrated. From this experiment alone, the choice of expression host is seen to influence the amount of recombinant protein produced. Piccolo® offers the scientist the capability to screen a large number of factors in one experiment. The optimum conditions can then be scaled up into a fermenter, and if a GFP fusion protein is used, the recombinant protein expression can be followed in the fermenter on-line in real-time using a fluorescence sensor.
This article is published under license to BioMed Central Ltd.