X-ray crystallography, structural determination

Low solubility or improper folding may sometimes hamper the use of enzymes, particularly when expressed in a non-native host. A method of expressing proteins with a C -terminal GFP fusion to use fluorescence as a measure of the amount of correctly folded protein has been introduced (44). DNA shuffling produced variants of ferritin that showed increased solubility, even when they were recloned without the GFP fusion. This assay has been used to produce proteins for X-ray crystallography structure determination (45). The protein nucleoside diphosphate kinase from Pyrobaculum aerophilum is insoluble when expressed in E. coli, but after DNA shuffling, a functional variant with six mutations was found to have 90% solubility, which enabled its crystallization, and its structure was determined. 

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