High-Throughput Pipeline for Protein Expression and Solubility Profiling Using Synthetically Generated Plasmids.

High-throughput protein expression and purification can now take advantage of the copious expansion of genes from extensive genomics and metagenomic surveillance programs. This article outlines a pipeline that involves strategic selection of a large repertoire of protein targets for cloning by commercial synthetic services, followed by high-throughput transformation, expression, and solubility screening. This pipeline will be applicable to structural and functional genomics approaches, as well as any project that requires large-scale screening for production of soluble proteins in a time-efficient manner. The protocols involve transformation, protein expression and solubility determination in a high-throughput fashion in a 96-well plate format. Proteins screened in this pipeline are then able to be purified for any assay needed. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Target optimization Basic Protocol 2: High-throughput transformation Basic Protocol 3: High-throughput expression and solubility screening.