Novelty:
A self-inducing highly efficient protein expression system for high-throughput functional proteomics.
Value Proposition:
To date, the use of functional proteomics for clinical applications, such as the monitoring of molecular biotoxins and identification of disease biomarkers, is limited by the inconvenient manual regulation of plasmid transcription and protein induction and insufficient protein yield of current expression systems, making them intractable for use in high-throughput methodology. Hence, this invention provides a temperature-sensitive auto-inducing expression plasmid for the efficient over-production of affinity-tagged Rop (repressor of primer) fusion proteins, enabling the large-scale expression and cost-efficient high-throughput screening of gene libraries. Advantages include:
• Simple, convenient auto-induction without temperature shifts, inducers, or specialized growth media
• Rapid isolation of high amounts of protein using the inbuilt purification tag and protease cleavage site
• Suitable for large-scale expression and high-throughput analysis of gene libraries
Technical Details:
Johns Hopkins researchers have engineered a temperature-sensitive plasmid for the self-inducing expression of affinity-tagged Rop-fusion proteins, suitable for large-scale expression and high-throughput functional proteomics. With technical advances leading to the availability of numerous complete genome sequences, there is now great interest in novel methods for the functional proteomics and expression profiling of large numbers of clinical samples. Hence, this invention created a temperature-sensitive copy number plasmid carrying a trp promoter upstream of the rop gene and its promoter, a repressor of plasmid DNA replication, for the generation of Rop-fusion constructs. Incubation of cells harboring the expression plasmid at 37C results in runaway replication and plasmid DNA accumulation to levels that greatly outnumber the genomic trp repressor molecules, thereby initiating protein expression without the need to monitor culture density or addition of inducers. Vector derivatives suitable for plasmid library construction, bacterial cells harboring the plasmid, as well as a kit comprising the expression vector are provided, and a purification tag and a protease cleavage site ensure the rapid protein isolation and analysis. Together, the ease of use and low cost of high-yield (340 mg per liter average yield and 1,300 mg per liter maximum yield) protein purification useful for high-throughput methodology make this technology a valuable new tool for the large-scale proteomic analysis of biological samples.
Looking for Partners:
To develop and commercialize the technology as a robust efficient tool for large-scale protein production suitable for high-throughput proteomic analyses.
Stage of Development: Pre-Clinical
Patent Status: Issued US 9284565
