Direct imaging of the recruitment and phosphorylation of S6K1 in the mTORC1 pathway in living cells

Knowledge of protein signalling pathways in the working cell is seen as a primary route to identifying and developing targeted medicines. In recent years there has been a growing awareness of the importance of the mTOR pathway, making it an attractive target for therapeutic intervention in several diseases. Within this pathway we have focused on S6 kinase 1 (S6K1), the downstream phosphorylation substrate of mTORC1, and specifically identify its juxtaposition with mTORC1. When S6K1 is co-expressed with raptor we show that S6K1 is translocated from the nucleus to the cytoplasm. By developing a novel biosensor we demonstrate in real-time, that phosphorylation and de-phosphorylation of S6K1 occurs mainly in the cytoplasm of living cells. Furthermore, we show that the scaffold protein raptor, that typically recruits mTOR substrates, is not always involved in S6K1 phosphorylation. Overall, we demonstrate how FRET-FLIM imaging technology can be used to show localisation of S6K1 phosphorylation in living cells and hence a key site of action of inhibitors targeting mTOR phosphorylation

Direct imaging of the recruitment and phosphorylation of S6K1 in the mTORC1 pathway in living cells

Knowledge of protein signalling pathways in the working cell is seen as a primary route to identifying and developing targeted medicines. In recent years there has been a growing awareness of the importance of the mTOR pathway, making it an attractive target for therapeutic intervention in several diseases. Within this pathway we have focused on S6 kinase 1 (S6K1), the downstream phosphorylation substrate of mTORC1, and specifically identify its juxtaposition with mTORC1. When S6K1 is co-expressed with raptor we show that S6K1 is translocated from the nucleus to the cytoplasm. By developing a novel biosensor we demonstrate in real-time, that phosphorylation and de-phosphorylation of S6K1 occurs mainly in the cytoplasm of living cells. Furthermore, we show that the scaffold protein raptor, that typically recruits mTOR substrates, is not always involved in S6K1 phosphorylation. Overall, we demonstrate how FRET-FLIM imaging technology can be used to show localisation of S6K1 phosphorylation in living cells and hence a key site of action of inhibitors targeting mTOR phosphorylation

Baculovirus as delivery system for gene transfer during hypothermic organ preservation

Concerns over the safety of conventional viral vectors have limited the translation of gene transfer from an exciting experimental procedure to a successful clinical therapy in transplantation. Baculoviruses are insect viruses, but have the ability to enter mammalian cells and deliver potential therapeutic molecules with no evidence of viral replication. This study provides evidence of the ability of recombinant baculovirus to enter mammalian kidneys and livers during cold preservation. Six kidneys and six liver lobules retrieved from large pigs were perfused with University of Wisconsin (UW) solution containing a baculovirus tagged with green fluorescent protein and preserved for 8 h. In addition, six kidneys were perfused with UW containing a baculovirus expressing red fluorescent protein and preserved for 24 h. Green fluorescent virus particles were detected within transduced kidneys and livers after 8 h standard cold storage and red fluorescent protein mRNA was detected in kidneys after 24 h of cold preservation. There were no significant differences in tissue architecture, cell morphology or ATP content between experimental organs and their controls. Ex vivo transduction of organs with recombinant baculovirus during conventional cold preservation was demonstrated with no evidence of additional injury or reduction in cell viability. © 2011 European Society for Organ Transplantation

Generation of baculovirus vectors for the high-throughput production of proteins in insect cells.

The baculovirus expression system is one of the most popular methods used for the production of recombinant proteins but has several complex steps which have proved inherently difficult to adapt to a multi-parallel process. We have developed a bacmid vector that does not require any form of selection pressure to separate recombinant virus from non-recombinant parental virus. The method relies on homologous recombination in insect cells between a transfer vector containing a gene to be expressed and a replication-deficient bacmid. The target gene replaces a bacterial replicon at the polyhedrin loci, simultaneously restoring a virus gene essential for replication. Therefore, only recombinant virus can replicate facilitating the rapid production of multiple recombinant viruses on automated platforms in a one-step procedure. Using this vector allowed us to automate the generation of multiple recombinant viruses with a robotic liquid handler and then rapidly screen infected insect cell supernatant for the presence of secreted proteins

GFP-Based expression screening of membrane proteins in insect cells using the baculovirus system

A key step in the production of recombinant membrane proteins for structural studies is the optimization of protein yield and quality. One commonly used approach is to fuse the protein to green fluorescent protein (GFP), enabling expression to be tracked without the need to purify the protein. Combining fusion to green fluorescent protein with the baculovirus expression system provides a useful platform for both screening and production of eukaryotic membrane proteins. In this chapter we describe our protocol for the expression screening of membrane proteins in insect cells using fusion to GFP as a reporter. We use both SDS-PAGE with in-gel fluorescence imaging and fluorescence-detection size-exclusion chromatography (FSEC) to screen for expression

GFP-Based Expression Screening of Membrane Proteins in Insect Cells Using the Baculovirus System.

A key step in the production of recombinant membrane proteins for structural studies is the optimization of protein yield and quality. One commonly used approach is to fuse the protein to green fluorescent protein (GFP), enabling expression to be tracked without the need to purify the protein. Combining fusion to green fluorescent protein with the baculovirus expression system provides a useful platform for both screening and production of eukaryotic membrane proteins. In this chapter we describe our protocol for the expression screening of membrane proteins in insect cells using fusion to GFP as a reporter. We use both SDS-PAGE with in-gel fluorescence imaging and fluorescence-detection size-exclusion chromatography (FSEC) to screen for expression

Improved expression of secreted and membrane-targeted proteins in insect cells.

Secretory and membrane-bound proteins are generally produced in lower amounts in insect cells compared with cytoplasmic and nuclear proteins. There may be many reasons for this, including degradation of recombinant proteins by proteases, competition for cellular resources between native and recombinant proteins, and physical blockage of the secretory pathways. In the present study, we describe the construction of a baculovirus in which chiA (chitinase) and cath (cathepsin) genes have been deleted and show improved recombinant protein expression using this vector. We confirmed the complete removal of both genes by PCR, restriction enzyme analysis and enzyme assays, and the modified virus DNA was shown to be stable in bacterial cells over multiple passages. A selection of recombinant genes were inserted into the double-deletion virus and their expression levels compared with recombinant viruses that had single or no gene deletions. In all instances, the double-deletion viruses showed greatly enhanced levels of protein production for both secreted and nuclear/cytoplasmic proteins. In summary, we have conclusively demonstrated the importance of this deletion vector for the high-level production of recombinant proteins