High-Throughput Expression Screening in Mammalian Suspension Cells.

Proteins naturally expressed in eukaryotic organisms often require host chaperones, binding partners, and posttranslational modifications for correct folding. Ideally the heterologous expression system chosen should be as similar to the natural host as possible. For example, mammalian proteins should be expressed in mammalian expression systems. However, this does not guarantee a protein will be expressed in a sufficient high yield for structural or biochemical studies or antibody generation. Often a screening process is undertaken in which many parameters including truncations, point mutations, investigation of orthologs, fusion to peptide or protein tags at the N- or C-terminus, the coexpression of binding partners, and even culture conditions are varied to identify the optimal expression conditions. This requires multiparallel expression screening in mammalian cells similar to that already described for E. coli expression. Here we describe in detail a multiparallel method to express proteins in mammalian suspension cells by transient transfection in 24-well or 96-well blocks.

Expression screening in mammalian suspension cells.

Proteins naturally expressed in eukaryotic organisms often require host chaperones, binding partners, and posttranslational modifications for correct folding. Ideally the heterologous expression system chosen should be as similar to the natural host as possible. For example, mammalian proteins should be expressed in mammalian expression systems. However this does not guarantee a protein will be expressed in a sufficient high yield for structural or biochemical studies or antibody generation. Often a screening process is undertaken in which many variants including truncations, point mutations, investigation of orthologues, fusion to peptide or protein tags at the N- or C-terminus, the co-expression of binding partners, and even culture conditions are varied to identify the optimal expression conditions. This requires multi-parallel expression screening in mammalian cells similar to that already described for E. coli expression. Here we describe in detail a multi-parallel method to express proteins in mammalian suspension cells by transient transfection in 24-well blocks.

Extended budded virus formation and induction of apoptosis by an AcMNPV FP-25/p35 double mutant in Trichoplusia ni cells.

An Autographa californica nucleopolyhedrovirus (AcMNPV) mutant (AcdefrT) isolated from virus-infected Trichoplusia ni (TN-368) cells produced plasma membrane blebbing and caspase-3-like activity late in infection. It also synthesized less polyhedra, but displayed enhanced budded virus formation in TN-368 cells. This phenotype resulted from dual mutations in p35 and FP-25. In this study we showed that enhanced budded virus production occurs because the hourly rate of release of virus from AcdefrT-infected cells is higher than that for AcMNPV and it continues for longer. This may be the trigger for the induction of apoptosis late in AcdefrT-infected TN-368 cells. However, laddering of host DNA was absent in TN-368 cells infected with AcdefrT, but was observed in Spodoptera frugiperda cells. Very late polyhedrin protein production and occlusion body formation was reduced in AcdefrT-infected TN-368 cells, but chitinase and capsid late gene expression remained unchanged. The AcdefrT was rescued with a copy of a baculovirus iap3, to replace the absent p35. This modification abolished most plasma membrane blebbing in AcdefrT-infected TN-368 cells, but did not affect enhanced budded virus production. These data suggest that inhibitors of apoptosis are required in T. ni cells, particularly when the production of budded virus is enhanced.

Multiplexed expression and screening for recombinant protein production in mammalian cells.

BACKGROUND: A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner. RESULTS: A nested PCR approach was used to clone 21 extracellular domains of mouse receptors as CD4 fusions within a mammalian GATEWAY expression vector system. Following transient transfection, HEK-293E cells grown in 2 ml cultures in 24-deep well blocks showed similar growth kinetics, viability and recombinant protein expression profiles, to those grown in 50 ml shake flask cultures as judged by western blotting. Following optimisation, fluorescent dot blot analysis of transfection supernatants was shown to be a rapid method for analysing protein expression yielding similar results as western blot analysis. Addition of urea enhanced the binding of glycoproteins to a nitrocellulose membrane. A good correlation was observed between the results of a plate based small scale transient transfection dot blot pre-screen and successful purification of proteins expressed at the 50 ml scale. CONCLUSION: The combination of small scale multi-well plate culture and dot blotting described here will allow the multiplex analysis of different mammalian expression experiments enabling a faster identification of high yield expression constructs or conditions prior to large scale protein production. The methods for parallel GATEWAY cloning and expression of multiple constructs in cell culture will also be useful for applications such as the generation of receptor protein microarrays.

Dual mutations in the Autographa californica nucleopolyhedrovirus FP-25 and p35 genes result in plasma-membrane blebbing in Trichoplusia ni cells.

Spodoptera frugiperda cells infected with Autographa californica nucleopolyhedrovirus (AcMNPV) lacking a functional anti-apoptotic p35 protein undergo apoptosis. However, such mutants replicate normally in Trichoplusia ni (TN-368) cells. An AcMNPV plaque isolate (AcdefrT) was identified during propagation of a virus deficient in p35 in TN-368 cells. This virus exhibited enhanced budded-particle formation in TN-368 cells, but was partially defective for polyhedra production in the same cells. Virus replication in AcdefrT-infected TN-368 cells was accompanied by extensive plasma-membrane blebbing and caspase activation late in infection, both features of apoptosis. Rescue of the p35 locus of AcdefrT continued to result in a reduction in polyhedra and increase in budded virus production in TN-368 cells, but no plasma-membrane blebbing was observed. The mutation was mapped to the FP-25 gene locus. This gene mutation combined with the non-functional p35 was found to be responsible for the cell-blebbing effect observed in AcdefrT-infected TN-368 cells.

Non-polar distribution of green fluorescent protein on the surface of Autographa californica nucleopolyhedrovirus using a heterologous membrane anchor.

Heterogeneous proteins can be displayed on the surface of the budded form of Autographa californica nucleopolyhedrovirus (AcMNPV) after fusion of the display protein to the AcMNPV major envelope glycoprotein, gp64. However, display is restricted to the poles of the virion and is relatively low level. To investigate the use of alternative membrane anchor sequences that would be compatible with virus surface display, we have constructed a display vector containing the gp64 signal peptide and a membrane anchor from the vesicular stomatitis virus (VSV) G glycoprotein. Introduction of a gene encoding green fluorescent protein (GFP) between these signals led to abundant display of GFP on the surface of insect cells and on recombinant budded virions. In addition, and in contrast to gp64 based fusion proteins, GFP was localized to the lateral virion surfaces.

Characterization of a truncated soluble form of the baculovirus (AcMNPV) major envelope protein Gp64.

A truncated tagged form of the Autographica californica multiple nuclear polyhedrosis virus major surface glycoprotein, gp64, has been expressed using the baculovirus expression system and purified to homogeneity by immune-affinity chromatography. The protein, which is responsible for virus-cell fusion, was a trimer in solution and retained this oligomeric form at pH 5, the pH of fusion. Circular dichroism spectroscopy indicated a protein with mixed alpha-helix and beta-sheet content that did not undergo significant change at pH 5. The soluble protein showed no detectable binding to the insect cell surface. These data suggest a novel fusion mechanism for gp64 compared to models such as the influenza HA. In a crystal screen, deglycosylated, but not glycosylated, preparations of the protein were found to form small needle-shaped crystals that may form the basis of a dedicated structural study.