Until recently, small scale expression trials in the baculovirus system were commonly carried out in small volume shake flasks or adherent cell cultures [6
]. While these formats have the advantage of simplicity and familiarity, it can be prohibitively cumbersome to process multiple samples in parallel, scale-up to larger production volumes can be inconsistent, and this approach is wasteful as most of the cell culture is not needed for downstream analysis [6
]. To obviate these problems, we developed the Vertiga-IM for growing small volumes of cell cultures in suspension. The key to the success of this instrument was careful optimization of the shaker’s orbital radius. If the throw-arm length was too small, cells were damaged by shear forces, whereas a throw-arm that was too large resulted in settling of the cells and suboptimal aeration (data not shown). In this particular instance, we utilized Sf9 insect cells for expression, which were maintained in suspension in 24-well blocks with a 1/2” throw arm at 300 rpm. The length of the throw arm and the rotation speed can be optimized for other cell types or culture vessels.
Development of protocols for baculovirus microexpression that use the Vertiga-IM was carried out using fourteen C-terminal truncation variants created from the full length β2AR gene. A parallel expression process was designed with this device in conjunction with the Guava EasyCyte microcapillary flow cytometer. The latter being used to assess cell surface targeting of heterologously expressed FLAG epitope tagged receptor (). Three truncations mutants were then chosen for scale up to medium scale shake flasks along with wtβ2AR for the purpose of comparing cell surface expression data to two other commonly used metrics: saturation ligand binding and protein immunoblot.
Results from medium scale expression studies showed high correlation between cell surface expression and the two more traditional metrics (), providing sufficient confidence so that cell surface expression data may be used as a means to screen out low expressing constructs in small scale expression trials prior to further characterization and scale up. We have further been able to use MFI of cell surface expression as a metric for predictive scale up in Wave bioreactors (data not shown). Although the correlation in positive expression data helps to select a construct for scale up and biophysical analysis, there are inconsistencies in the data (e.g. expression levels and Bmax values) that may be due to a higher limit of detection for the FACS analysis or a difference between the amount of functional protein being produced and the amount of total protein trafficked successfully to the cell surface. Indeed, we have observed an apparent early saturation of cell surface expression levels for other constructs in this system, while total functional protein levels continue to increase. This problem is much more pronounced when the target is expressed under the polyhedrin late stage promoter, rather than the GP64ie1 fusion promoter (Novagen) presumably due to the adverse effect of viral infection on the endogenous secretory machinery of the cell. Nevertheless, the ability to screen constructs in a high-throughput manner not only conserves reagents, but may also eliminate the need to generate high-titer stocks of each construct being screened, as the P2 viral passages may contain enough virus under favourable conditions to infect small scale expression cultures for screening purposes.
In our second case study of human Cx26, the cytoplasmic orientation of the N- and C-tails precluded the use of the cell surface expression assay, and there is no straightforward method to quantitatively measure channel activity. Therefore, we used the Vertiga-IM for parallel expression in a Thomson 24-well plate format, monitoring the expression level using semi-quantitative protein immunoblot analysis. The expression level varied greatly with the location and extent of truncation (). In general, truncations of the termini and the E1 extracellular loop are not viable or result in much lower protein expression. However, truncations of the IL and E2 loops were surprisingly well tolerated and therefore reasonable to pursue further. This study has provided a large number of potentially useful constructs, as well as important guidance to where and to what extent truncations can be made to the molecule in the future.