A High-Throughput Multi Well Plate-Based Approach for the Combined Expression, Export, and Assay of Recombinant Proteins.

High-throughput screening (HTS) of proteins is used in a wide range of applications across the biology, biotechnology, and medicine disciplines. These include yield optimization, 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 assays), 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 enough 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 Escherichia coli cells in the same microplate well. 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 medium. The resulting protein is of sufficient purity and yield that it 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. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Expression, export, and isolation of vesicular-packaged recombinant protein Support Protocol 1: 96-well plate cold-shock transformation Support Protocol 2: In-plate affinity-tag protein purification Support Protocol 3: Example in-plate enzymatic assay.