A High-Throughput Multiwell-Plate based Approach for the combined Expression, Export and Assay of Recombinant Proteins.

High-throughput screening (HTS) of proteins has a wide range of applications across the biology, biotechnology, and medicine disciplines. These include yield optimisation, drug or biomarker discovery, and protein engineering, among others. Factors that need to be considered in designing high throughput protein expression and screening methods, be that for expression, activity, stability, or binding as says, include the required yield, reproducibility, solubility, stability, purity and activity of the protein. Thus, larger culture volumes and time-consuming manual protein extraction and purification steps are normally required to produce sufficient quantity of protein of appropriate purity. This limits the type of assay, and number of protein variants that can be simultaneously tested in an experiment. Here we describe a HTS protocol that allows the overnight expression, export and assay of recombinant proteins from E. coli cells in the same multi-well plate tube. The protocol uses a recently described Vesicle Nucleating peptide (VNp) technology that promotes high yield vesicular export of functional proteins from E. coli into the culture media. The resulting protein is of sufficient purity and yield that in can be used directly in plate-based enzymatic assays without additional purification. This simple single plate protocol allows itself to a wide range of high-throughput research and development screening applications, ranging from streamlining protein production and identification of activity enhancing mutations, to ligand screening for basic research, biotechnological and drug discovery applications.