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Soluble proteins, with high expression levels, are preferred candidates for structural and functional studies. In cases of low expression, aggregation or inclusion body formation, time-consuming searches for optimal expression or refolding conditions are required. We have developed a high-throughput solubility engineering and screening platform for proteins that are expressed in an insoluble form in  Escherichia coli  with the aim of obtaining a broad spectrum of best hits with increased solubility in difficult to express target proteins. This process has been developed using error-prone PCR to introduce random base changes in genes of interest. Expression of mutated proteins in fusion with the reef coral fluorescent protein ZsGreen as a solubility marker has enabled the selection of more soluble variants. We have used a colony picker to achieve high-throughput selection of  E.coli  expressing more soluble target protein–ZsGreen fusions, with increased fluorescence. The whole process enables us to complete one round of mutation, screening and analysis of 20 000 potential soluble clones within ∼8 weeks. We describe the development of the methods using different model proteins and show one example, the kinase domain from the human EphB2 receptor, as a successful application of the whole platform.

Development of a screening platform for directed evolution using the reef coral fluorescent protein ZsGreen as a solubility reporter