Life Science
by Imre Berger, Maxime Chaillet, Frederic Garzoni, Sinyee Yau-Rose, and Barney Zoro
High-Throughput Screening of Multiple Protein Complexes
K
ey protein complexes are fundamental to a wide range of cellular processes and key metabolic pathways within the body. Their dys- function is associated with a range of disease pathologies such as cystic fibrosis, Alzheimer’s disease, and Parkinson’s disease.1,2
cultures require the temperature to be maintained within 25–28 o The study of
the molecular structure and function of multiprotein complexes is there- fore of value for the successful determination of their role in biological pathways and for the development of diagnostics and therapeutics. How- ever, for this to be possible, techniques are needed that can increase both the yield of protein and the number of complexes that can be produced.
This article describes an automated method for optimizing expression of multiprotein complexes in insect cells using the advanced microscale bioreactor (ambr™) system (TAP Biosystems, Royston, U.K.). The applica- tion of laboratory automation can facilitate medium- to high-throughput multicomplex protein expression programs and, when combined with good experimental design, shows that protein expression can be rapidly improved compared to the traditional shake flask method.
Study of eukaryotic multiple protein complexes For successful molecular and structural studies of protein complexes, it is
necessary to obtain large quantities of protein. However, the low abun- dance of these endogenous protein complexes, as well as their inherent instability following successful isolation, commonly inhibit molecular and structural studies. Advances in molecular biology and the use of recombi- nant biology techniques over the past decade for the overexpression of such proteins have been invaluable. Following the identification and isola- tion of the genetic material encoding these protein complexes, there are a host of prokaryotic and eukaryotic systems available for their expression and synthesis.3
Currently the most widely employed systems for the isolation and study of recombinant human multiprotein complexes are baculovirus expression vector systems (BEVS), which employ insect cell lines as the host cell for protein expression. This system provides the eukaryotic host machinery necessary to support correct post-translational modification for authentic processing, with correct structural conformation and activity of the pro- tein complex. With the ability to insert multiple genes into the baculovirus vector, infected insect cell lines can be established that express multiple protein subunits and hence multiprotein complexes. BEVS also enable in situ engineering of individual protein components, making it possible to express a wide range of protein complex variants.
In order to establish, grow, and maintain an insect cell culture in suspension, specific oxygen, osmolality, nutrient, pH, and temperature are required. Continual monitoring of growth conditions is necessary to ensure suc- cessful infection and recombinant protein overexpression. Insect cell line
C, and it
is essential that insect cells are in log phase at the time of infection. In order to ensure cells are adequately oxygenated and remain in suspension, tradi- tional methods that use shake or spinner flasks are commonly employed.
Issues with recombinant expression systems for
multiprotein complexes BEVS such as the MultiBac, developed by the Berger laboratory at the European Molecular Biology Laboratory (EMBL, Grenoble, France), are widely used for the expression of large eukaryotic complexes with many genes encoding subunits.4
The MultiBac system enables in situ gene
manipulation and the study of multiple variants for screening purposes. Not only can the simultaneous establishment, growth, and study of large numbers of protein variants following infection with multiple viral clones prove costly and time consuming, but also pose difficulties for manual handling and monitoring.
Culturing dozens of protein variants in parallel demands adequate space in order to house large shaker platforms. The use of traditional shake flasks for such studies requires considerable reagent volumes and manual labor for experimental setup, continual monitoring of growth conditions, and sub- sequent protein expression studies. Furthermore, as with all experiments involving large sample numbers, manual error is inevitable. In summary, the use of shake flask systems does not readily provide the flexibility for the simultaneous study of a large number of altered recombinant multiprotein complexes and cell line variants in a screening program.
Figure 1 – ambr automated micro bioreactor system. AMERICAN LABORATORY • 32 • SEPTEMBER 2013
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