Development of a screening platform for directed evolution using the reef coral fluorescent protein ZsGreen as a solubility reporter.
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 approximately 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.
Heddle
Catherine
C
Global Protein Science and Supply, AstraZeneca R&D, Södertälje, Sweden. catherine.heddle@astrazeneca.com
Mazaleyrat
Sabine L
SL
D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D036183:Receptor, EphB2 / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012995:Solubility
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D036183:Receptor, EphB2