# A High‐Throughput Multi Well Plate–Based Approach for the Combined Expression, Export, and Assay of Recombinant Proteins

## TL;DR

This paper introduces a high-throughput method for expressing, exporting, and testing recombinant proteins in a single microplate well using E. coli and a new Vesicle Nucleating peptide technology.

## Contribution

A novel single-plate protocol for high-throughput protein expression, export, and assay using vesicle-based purification in E. coli.

## Key findings

- The protocol enables overnight expression and export of functional proteins directly into the culture medium.
- Proteins produced are pure enough for direct use in enzymatic assays without additional purification.
- The method supports a wide range of high-throughput screening applications in biotechnology and drug discovery.

## Abstract

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