A Portable Vector Maximizing Protein Expression in High-Density Bacterial Cultures Using Either Temperature or Chemical Induction

Background Bacterial fermentation remains a popular and reliable form of protein production for both research and commercial applications. Current systems require complementary functional elements present in both engineered bacterial strains and plasmid expression vectors to insure tight control on gene expression. Expression control is important to obtain the best yield and highest functional activity of protein products.

However, commercially available, or stock, bacterial strains and plasmid vectors often do not provide for acceptable expression in terms of the yield, subcellular localization, solubility, folding consistency and activity of the protein products. The reliance of plasmid vectors on particular genetic features in the production strains prevents most vectors from being portable and testable in a wider range of production host genotypes. The limitations of the existing commercially available systems leave researchers with relatively few options if the proteins of their interest are poorly expressed.

Invention Description Researchers at The University of Texas at Austin have recognized the limitations of present production systems and have devised an innovative plasmid expression vector that maintains tight control on heterologous protein expression, yet is functional in wide range of bacterial genotypes. This expression plasmid brings the benefits of a high-copy plasmid that can be induced both by temperature or addition of a non-toxic chemical compound.

Mild temperature increase from 30° to 37°C leads to potent stimulation of gene expression without co-induction of heat shock responses, which often have deleterious effects on the folding properties or aggregation state of the protein product. Alternatively, chemical induction methods can be employed at temperatures less than 37°C leading to high product accumulation and lessened host response.

This plasmid system is compatible with a range of recA+ and recA- bacterial genotypes. The plasmid's portable nature allows the researchers, not a catalog, determine the most appropriate bacterial strain for production. Further, using this system, dramatically increased protein yields are observed due to the ability to induce expression in bacterial cultures that are ten times denser than standard expression systems and induction periods that can extend twice as long as standard systems. These characteristics make this plasmid readily adaptable to high-density, batch-fed production processes. Finally, this system has been shown to allow the production of several large and complex proteins at high yields (several percent of total cellular protein).

Development Stage Lab/bench prototype

IP Status One PCT patent application filed

UT Researcher Kenneth A. Johnson, Ph.D., Chemistry and Biochemistry, The University of Texas at Austin John W. Brandis, Ph.D., ICMB, The University of Texas at Austin

Type of Offer: Licensing

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