Research
CSBMP Research
The Specific Aims for the research to be conducted within the center and the participating investigators are:
- To analyze the specific role of particular lipids in membrane protein structure and assembly using Rhodobacter sphaeroides as a model system. By modifying the lipid composition in vivo, the impact of lipids on the purification, activity, crystallizability, and diffraction quality can be assessed using the membrane protein complex cytochrome c oxidase. A newly developed mass spectrometry method will enable the detailed analysis of lipid content of purified and crystallized protein.
Ovine prostaglandin H synthase
Participating Investigators:
Shelagh Ferguson-Miller - R. sphaeroides expression methodologies
Christoph Benning - Methodologies for genetically altering the lipid composition of R. sphaeroides membranes -
To explore in depth different expression systems (mammalian cell, insect cell, bacteria, yeast) and to develop strategies for their optimization. Several expression systems have been successfully used at MSU to produce membrane proteins for structural analysis. However, criteria for selecting the “best” system to use for a particular target are almost nonexistent or are very subjective. Strategies to optimize or expand the usefulness and production capacity of expression systems are also limited. By examining their performance with a wide variety of proteins from the collaborator group, we intend to assess where the primary limitations are for membrane protein expression.
Participating Investigators:
R. Michael Garavito - Expression methodologies for E. coli.
David DeWitt; Development of new methodologies for high-yield membrane expression in mammalian cells
Suzanne Thiem - Development of new methodologies for high-yield membrane expression in insect cells
Andreas Weber and R. Michael Garavito; Expression methodologies for yeast. -
To expand existing methodologies for high-yield membrane protein expression using fermentors and bioreactors. Extensive observations from many groups reveal that membrane proteins can be expressed at modest levels, but that attempts to increase the scale of production is often not possible. Experiments are under way to elucidate the factors that can increase cell and membrane protein yields using high-density fermentation. By optimizing efficiency of the expression vector and the protocols for high-density fermentation in tandem, the maximum yield of active membrane protein target can be achieved.
Participating Investigators:
R. Michael Garavito - Expression vector design for high-yield membrane protein expression in fermentors.
R. Mark Worden - Development of new bioproscessing methods for high-yield membrane protein expression in fermentors.
Allied Research Efforts
The Protein Expression Laboratory (Director: R. Mark Worden)
In Engineering at MSU has resources for computer-controlled fermentation at the 1-10 liter scale, as well as a 100 liter fermenter to be operational in early 2005. PEL personnel are available to aid investigators in large-scale expression of proteins.
Genome Sequencing of Galdieria sulphuraria (NSF EF-0332882; PI: A. Weber)
The red micro-alga G. sulphuraria (Cyanidiales) is a unicellular and extremophilic eukaryote that is adapted to living in hot sulfur springs (pH 0.05 to 4; to 56°C). The thermo-acidophilic eukaryote represents a particular interesting species for a structural genomics and proteomics approach owing to its extraordinary metabolic versatility such as heterotrophic and mixotrophic growth on more than 50 different carbon sources. It also tolerates high concentrations of toxic metals such as cadmium, mercury, aluminum or nickel. What allows this organism to survive in such extreme conditions are the numerous membrane transporters. G. sulphuraria was chosen as a sequencing target not only because its transporters are interesting from a biotechnology standpoint, but also their thermostability make them excellent targets for high resolution structural analysis. An EST library and high-throughput genomic sequence reads covering > 70% of the G. sulphuraria genome are now publicly available and candidate genes are being identified whose gene products are highly homologous to mammalian membrane proteins.
Center for Nanostructured Biomimetic Interfaces (Director: R. Mark Worden)
CNBI is comprised of 11 researchers having expertise in molecular biology, protein expression, separation science, polymer chemistry, interfacial science, nanotechnology, microelectronics, medicine, and commercial sensing technologies. The mission of the CNBI is to develop novel biomimetic interfaces that have active biological systems reconstituted into artificial biomembranes assembled on electrode surfaces. These interfaces will then be used to fabricate novel devices of commercial significance, including biosensors and biocatalytic systems. The specific research objectives of the current projects are to:
- Express and purify recombinant dehydrogenase enzymes, Major Facilitator Subfamily (MFS) transporter proteins, and gated ion channels;
- Develop novel classes of functional biomimetic interfaces containing dehydrogenase enzymes, Major Facilitator Subfamily (MFS) transporter proteins, and gated ion channels.
- Apply a variety of physical, electrical, and optical methods to measure, and optimize performance properties of the biomimetic interfaces.
- Develop practical devices and processes based on the biomimetic interfaces, including biosensor arrays, for applications in medical and pharmaceutical research.