Efficient Microfluidic Screening Method Using a Fluorescent Immunosensor for Recombinant Protein Secretions

Microbial secretory protein expression is widely used for biopharmaceutical protein production. However, establishing genetically modified industrial strains that secrete large amounts of a protein of interest is time-consuming. In this study, a simple and versatile high-throughput screening method for protein-secreting bacterial strains is developed. Different genotype variants induced by mutagens are encapsulated in microemulsions and cultured to secrete proteins inside the emulsions. The secreted protein of interest is detected as a fluorescence signal by the fluorescent immunosensor quenchbody (Q-body), and a cell sorter is used to select emulsions containing improved protein-secreting strains based on the fluorescence intensity. The concept of the screening method is demonstrated by culturing Corynebacterium glutamicum in emulsions and detecting the secreted proteins. Finally, productive strains of fibroblast growth factor 9 (FGF9) are screened, and the FGF9 secretion increased threefold compared to that of parent strain. This screening method can be applied to a wide range of proteins by fusing a small detection tag. This is a highly simple process that requires only the addition of a Q-body to the medium and does not require the addition of any substrates or chemical treatments. Furthermore, this method shortens the development period of industrial strains for biopharmaceutical protein production.

Automated summary

In this study, a simple and versatile high-throughput screening method for protein-secreting bacterial strains is developed using microemulsions. The secreted protein is detected using a fluorescent immunosensor and selected using a cell sorter. This method was successfully used to screen and improve the secretion ability of Corynebacterium glutamicum strains, increasing the secretion of fibroblast growth factor 9 threefold compared to the parent strain. This screening method can be applied to a wide range of proteins by fusing a small detection tag, and it shortens the development period of industrial strains for biopharmaceutical protein production.