SBIR-STTR Award

Robust expression of multi-subunit protein complexes is crucial for understanding the basic biology of organisms. Expression of the individual components in E. coli or yeast may be quick and cost-efficient, but many mammalian proteins show poor expression or solubility when expressed in these hosts. Further, they lack appropriate post-translational modifications, which may be essential for proper activity or interaction with other proteins. Current mammalian expression systems typically express one protein per plasmid, maintained either in the cytoplasm or randomly integrated onto the chromosome. Stable and coordinated expression of these proteins is not possible. Further complications arise in monitoring and analyzing complexes formed from the recombinant proteins. The goal of this Phase I proposal is therefore to develop a system to conditionally express multiple proteins as a mammalian operon on a stable "mini chromosome". The expression vector will be based on the linear "pJAZZ" vector, which removes functional and structural barriers to cloning. In addition, we will develop novel bacterial phytochromes (PHYs) as red and near-infrared fluorophores to monitor and quantify transient interactions among proteins. These PHYs will be useful for fluorescent imaging in vivo and in denaturing gels. Moreover, they will have FRET capability. This system will enable a wide range of studies on structure, function, assembly, and interaction of proteins in multi-subunit complexes. It will also facilitate identification of binding partners and improved imaging of cellular structures.

Public Health Relevance:
A large number of proteins within cells form unique multi-subunit complexes, which are typically very difficult to study. We propose to develop a novel system to readily produce and monitor protein complexes in cell cultures. This work will enable researchers to better understand protein interactions in normal and disease situations.

Public Health Relevance Statement:
A large number of proteins within cells form unique multi-subunit complexes, which are typically very difficult to study. We propose to develop a novel system to readily produce and monitor protein complexes in cell cultures. This work will enable researchers to better understand protein interactions in normal and disease situations.

NIH Spending Category:
Biotechnology; Genetics; Human Genome

Project Terms:
3-Dimensional; base; Binding (Molecular Function); Biological Assay; Biology; C-terminal; Cell Culture Techniques; Cell membrane; Cells; Cellular Structures; Chromosomes; Cloning; Code; Codon Nucleotides; commercialization; Complex; cost; Cytoplasm; Detection; Development; Disease; DsRed; Escherichia coli; Evaluation; expression vector; Fluorescence; Fluorescence Resonance Energy Transfer; fluorophore; Gel; Gene Proteins; Genes; Goals; Government; Green Fluorescent Proteins; Human Herpesvirus 4; Image; Image Analysis; improved; in vivo; Individual; Libraries; light scattering; Maintenance; Messenger RNA; Monitor; mutant; N-terminal; novel; Nuclear; Operon; Oranges; Organism; Oxygen; Peptide Signal Sequences; Peptides; Persons; Phase; Phytochrome; Plasmids; Post-Translational Protein Processing; Promotor (Genetics); Property; protein complex; Protein Complex Subunit; protein expression; protein protein interaction; Proteins; Recombinant Proteins; Recombinants; Replication Origin; Reporter; Research; Research Personnel; Sampling; Signal Transduction; Small Business Innovation Research Grant; Solubility; Spectrum Analysis; Structure; System; Testing; Time; Tissues; tool; Variant; vector; Viral; VP 16; Work; Yeasts

The proteins encoded by the human genome may participate in as many as 200,000 pairwise protein interactions, and “nearly every major process in a cell is carried out by assemblies of 10 or more interlocking protein molecules” (Bruce Alberts, Cell 1998). However, there are no commercially available expression systems for regulated expression of multi-subunit protein complexes. There is an acute need for a mammalian expression system that can produce multi-protein complexes, is tightly regulated, stable in prolonged culture, and non-integrated. No reported expression system comes near to meeting all these needs. Lucigen will develop and commercialize a stable episomal expression system with unbiased cloning ability, tunable light-inducible promoters, improved fluorescent tags, and the ability to express equivalent amounts of multiple proteins. This system will facilitate direct experimental validation of the myriad protein complexes predicted by computational models, as well as those that are already known (e.g, ion channels, multi-subunit enzymes, biochemical pathways).

Public Health Relevance Statement:
This Phase II research will result in a novel mammalian expression vector, light inducible promoters, new fluorescent proteins for high-throughput assays, and stable cell lines that will greatly facilitate cloning, expression, and high throughput screening for numerous research and pharmaceutical applications. The products developed in this proposal will be critical for functional analysis of the human protein “interactome” in normal and diseased states. This knowledge will inform and confirm computational and in vitro models, enabling critical advances in basic research and development of therapeutics.

Project Terms:
abstracting; Acute; Angiogenesis Modulating Agents; arm; base; Basic Science; Biochemical Pathway; Biological Assay; Cell Culture Techniques; Cell Line; Cells; Characteristics; Chromosomes; Cloning; Cloning Vectors; commercialization; Complementary DNA; Complex; Computer Simulation; design; Development; DNA Sequence; Elements; Energy Transfer; Enzymes; Episome; Escherichia coli; Excision; expression cloning; expression vector; Extinction (Psychology); Feedback; Fluorescence; Funding; Gene Expression; Genes; Genetic; Goals; Growth Factor; high throughput screening; Human; Human body; Human Genome; improved; In Vitro; in vitro Model; in vivo; Individual; Ion Channel; Knowledge; Libraries; Light; Mammalian Cell; meetings; Methods; Modeling; Monitor; mRNA Decay; Mutagenesis; Mutate; Mutation; Neurons; novel; Operon; Pattern; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Process; Promotor (Genetics); Protein Analysis; protein complex; Protein Complex Subunit; protein expression; protein protein interaction; Proteins; quantum; Regulation; Reporter; Reporting; Research; research and development; Research Personnel; Scientific Advances and Accomplishments; Shuttle Vectors; Small Business Innovation Research Grant; stable cell line; Staging; synthetic biology; System; Therapeutic; tool; Transfection; tumor; Tumor Suppression; Validation; Variant; vector; vi