Expression Laboratory
Funding ended: 31 December, 2001
Regular users of the expression laboratory:
Permanent:
Martin Svenda (Biochem, UU)
Student: Elin Pless (GenPat, UU)
Fredrik Söderbom (MolBiol, SLU)
Student: Andrea Hinas (MolBiol, SLU)
Post doc: Anders Aspegren (MolBiol, SLU)
Talal Gariani (ICM, UU)
Diane Lester (MolBiol, SLU)
Frequent:
Alasdair MacKenzie-Hose (MolBiol, SLU)
Jenny Berglund (MolBiol, SLU)
Anton Zharkov (MolBiol, SLU)
Saeid Karkehabadi (MolBiol, SLU)
Martin Svenda:
- Construction of a general platform to display a protein of choice in an ordered and regular manner:
Status: Progress has been made and I now have a platform that meet some of the requirements but further development of the possible pH range for binding is ongoing.
- Cloning, expression and purification of proteins involved in Clavaminic acid
synthesis and Cephamycin C synthesis in Streptomyces Claviligerus:
This project is part of an EU collaboration on creating new antibiotics. The purpose is to determine the structure of as many proteins involved in these gene clusters as possible in order to simplify and expand the possibilities of producing new antibacterial compounds.
Status: A number of genes from both clusters have been cloned into various expression systems for overexpression. Purification schemes have been developed for some of the expressed proteins. The regulatory proteins claR and CcaR have gone to crystallisation trials.
- Cloning, expression and crystallisation of Bestrophin.
(In collaboration with Claes Wadelius, Dept Genetics and Pathology, UU)
Best vitelliform macular dystrophy, an early onset degeneration of the macula of the patient, is caused by mutations in the gene for the protein Bestrophin. The function of the protein is not known but preliminary results indicate a role for the protein in intracellular signalling. To get insight into the function of the protein, the gene has been cloned into various expression vectors to be used in protein-protein interaction studies as well as for overexpression of the protein for crystallisation.
Status: A system for overexpression and purification of the C-terminal domain of the protein have been developed. Crystallisation trails ongoing. A system for overexpression and purification of the complete, membrane bound Bestrophin in Pichia Pastoris is under development. A scheme to find proteins interacting with Bestrophin has been initiated, using techniques such as GST-pulldown, co-immunoprecipitation and far-western.
-Cloning, expression and purification of proteins involved in the replication and transcription of a paramyxovirus.
(In collaboration with Mikael Berg Dept. Vet. Microbiol/virol. SLU)
Paramyxoviruses cause a number of severe diseases, to mention only one, human respiratory syncytial virus is the main infant killer of the world today. Also, a number of new paramyxoviruses have emerged in the recent years, causing several widespread epidemics. The aim with this project is to overexpress and determine the structure of proteins involved in replication and transcription of the paramyxovirus LPMV.
Status: Overexpression and purification schemes for the NP,P and V proteins have been developed. Crystallisation trials ongoing. A scheme to find proteins interacting with the V protein (interfering with interferon production in infected cells) has been initiated, using techniques such as GST-pulldown, co-immunoprecipitation and far-western. Several possible candidates have been found.
Fredrik Söderbom:
Dictyostelium discoideum
My research interest focus on the study of genes/gene products that are involved in regulation of development of the lower eukaryotic organism Dictyostelium discoideum.
Dictyostelium habitats the forest floors where it feeds on bacteria. Upon starvation the single cell amoebae starts producing and secreting cAMP that acts as a signal for onset of development. In response to cAMP about 100 000 cells starts migrating towards an aggregation centre where they subsequently form an aggregate. The cells continue to develop and the cells start to diverge into two main cell types, pre-stalk and pre-spore cells. The final stage of development is the formation of a fruiting body where pre-stalk cells have developed into dead stalk cells that are topped by a ball of spores.
The genome, 34 Mb, is presently being sequenced and databases for homology searches are available.
Why Dictyostelium?
The advantages of using Dictyostelium as a model for developmental studies are numerous. The cells can be grown on bacteria or in liquid media with a generation time of 4 and 8 hours, respectively. Dictyostelium cells develop synchronously in the lab and go through the developmental cycle within 24 hours.
Most of the molecular tools available for yeast but not for higher organisms, can be applied on Dictyostelium. This gives us a handle to study biological processes, e.g. development, which lower cells lack and are difficult to study in higher eukaryotes.
Many of the signal transduction pathways in Dictyostelium are basically identical to those in higher eukaryotes, e.g. man. This fact emphasises the advantage of Dictyostelium as a model for developmental studies.
Projects
Post-transcriptional regulation
The regulation of mRNA levels in cells are of uttermost importance. It is in part controlled by gene transcription, which has been studied to a great extent. Another level of regulation is acting on the RNA after transcription, i.e. post-transcriptional control. This important process has recently acquired an increasing interest and many of the factors involved in the process have been analysed in yeast. Due to the lack of sophisticated molecular tools, the knowledge of the mechanisms that are involved in post-transcriptional regulation in higher eukaryotes is scares.
Defects in post-transcriptional control have turned out to be important for the onset of different diseases such as cancer.
Presently we are isolating different ribonucleases from Dictyostelium based on homology search of the genome, using known RNase sequences from other organisms. The isolated RNases will be knocked-out and the effect on development will be studied. Over-expression of the RNases will give us material for structural and mechanistic studies.
In addition we will screen for new RNases as well as for their mRNA targets by using microarray technique.
Small regulatory RNAs
Recently newly discovered small regulatory RNAs have generated a huge interest. These RNAs are involved in different processes such as splicing, ribosome biogenesis, telomere synthesis, RNA editing and have also been suggested to be involved in human diseases.
We have recently started a project to screen for such RNAs in Dictyostelium. Our main focus will be to isolate and characterise small RNAs involved in different developmental processes. The RNAs are presently being cloned and sequenced. Their function and structure will subsequently be investigated.
Protein degradation
The proteasome is a complex of different proteins involved in protein degradation. It seems to be ubiquitous in all eukaryotes. One of the members of the complex is PA28 which has been implied to target certain proteins for degradation.
We found a homologue of the protein by searching the Dictyostelium genome and isolated the gene. We could show that its activity is similar to that in higher eukaryotes. We are presently knocking out the gene in order to investigate its effect on development.
Diane Lester:
Diane Lester has cloned, expressed and purified a number of plant 2-oxoglutarate-dependent dioxygenases. The viability of refolding insoluble protein from inclusion bodies was tested in some cases. Crystallisation trials are underway for those that gave good yields of proteins. A region that has a central role in substrate binding in these enzymes was identified using site-directed mutagenesis.
Alasdair MacKenzie-Hose
Initial usage of the expression lab was concerned with the expression of recombinant plant oxidase genes in E. coli, making use of protein purification equipment in the lab. Furthermore, various plasmid constructs of the plant oxidases were constructed with and without affinity tags, and the resulting proteins screened for crystallisability.
I also assisted Dr David van der Spoel and his summer students with the production of recombinant protein of the HIV capsid protein for both crystallisation trials as well as dynamic light scattering measurements to verify the action of an inhibitor in its ability to prevent capsid oligomerisation.
Most recently my activities in the lab have focused on the cloning, expression and purification of enzymes involved in Clavaminic acid synthesis, as part of a EU collaboration on creating new antibiotics. One of the enzymes is through to crystallisation trials with promising results, while efforts are being made to get the other enzymes expressed at a level suitable for crystallisation trials.
Latest update at 29 March, 2001.