Figure 5 – SDS-PAGE and Western blot analysis of protein produced at CFY/YFP protein expression plateau (day 4). The target His-tagged 2-protein com- plex was positively identified against marker M (Precision Plus Protein™ All Blue Standards, Bio-Rad, Hercules, CA) and UCC (uninfected cell control).
culture parameters for high-throughput screening studies. With the ability to employ smaller culture volumes and monitor up to 24 or 48 bioreactors simultaneously, the automated system is well suited for the culture and study of multiple variants for rapid multiparallel protein expression.
High-throughput screening
studies of multiprotein complexes The study of large numbers of protein complex variants in parallel using manual shake flask methods is not only time consuming and expensive, but difficult to handle and evaluate. The automated workstation provides the ability to carry out high-throughput multiprotein expression screening studies, with minimal cost, time, and manual effort.
With the ability to simultaneously study up to 24 or 48 bioreactors per workstation employing low volumes of culture media per study, ambr enables multiple cell line selection and development experiments to be run simultaneously. This facilitates a wide range of parameter studies, such as the infectivity of viral clones, protein expression, and culture conditions in order to optimize insect cell protein expression and production. In ad- dition, the system can be used to study the stability and productivity of expressed protein complexes across a range of clones in order to select the most promising variants before scaleup, or to study multiple replicates of the same clone to compare the effect of different feeding strategies or culture conditions on growth and expression levels.
In summary, this streamlining of protein expression studies using auto- mated micro bioreactor systems will ultimately accelerate research on the structure and function of multiprotein complexes. The next step is to optimize the process to mimic larger-scale bioreactor cultures, as has successfully been achieved for mammalian cell cultures.6
development of multiprotein diagnostics or protein therapeutics in future drug discovery programs.
References 1. Guerriero, C.J.; Brodsky, J.L. The delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology. Physiol. Rev. 2012, 92(2), 537–76.
2. Andre, R.; Tabrizi, S.J. Misfolded PrP and a novel mechanism of protea- some inhibition. Prion 2012, 6(1), 32–6.
3. Rai, M.; Padh, H. Expression systems for production of heterologous proteins. Current Sci. 2001, 80, 1121–8.
4. Berger, I.; Fitzgerald, D.J. et al. Baculovirus expression system for het- erologous multiprotein complexes. Nat. Biotechnol. 2004, 22(12), 1583–7.
5. Chaillet M.; Garzoni, F. et al. MultiBac Expression System: comparison of growth and multiprotein production in shake flask and automated miniature bioreactor (ambr™) cultures. Poster presentation at ISBio- Tech 3rd Annual Meeting, Rosslyn, VA, Mar 11–15, 2013. 6. Hsu, W.T.; Aulakh, R.P. et al. Advanced microscale bioreactor system: a representative scale-down model for bench-top bioreactors. Cytotech- nology 2012, 64(6), 667–78.
Such studies are
not only of interest for the understanding of the mechanisms and the role of these proteins in biological pathways, but also for the potential
Dr. Imre Berger is Group Leader, Structural Biology of Eukaryotic Complexes and Head of the Eukaryotic Expression Facility (EEF); Maxime Chaillet is Engi- neer for HT Technologies, EEF; and Frederic Garzoni is Facility Manager, EEF, European Molecular Biology Laboratory (EMBL), Grenoble Cedex 9, France. Dr. Sinyee Yau-Rose is a Bioprocess Product Specialist, and Dr. Barney Zoro is ambr Product Manager, TAP Biosystems, York Way, Royston, Herts SG8 5WY, U.K.; tel.: +44 (0) 1763 227200; e-mail: barney.zoro@
tapbiosystems.com.
AMERICAN LABORATORY • 35 • SEPTEMBER 2013
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