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Engineering soluble proteins for structural genomics
Authors:Pédelacq Jean-Denis  Piltch Emily  Liong Elaine C  Berendzen Joel  Kim Chang-Yub  Rho Beom-Seop  Park Min S  Terwilliger Thomas C  Waldo Geoffrey S
Institution:Bioscience Division, MS-M888, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
Abstract:Structural genomics has the ambitious goal of delivering three-dimensional structural information on a genome-wide scale. Yet only a small fraction of natural proteins are suitable for structure determination because of bottlenecks such as poor expression, aggregation, and misfolding of proteins, and difficulties in solubilization and crystallization. We propose to overcome these bottlenecks by producing soluble, highly expressed proteins that are derived from and closely related to their natural homologs. Here we demonstrate the utility of this approach by using a green fluorescent protein (GFP) folding reporter assay to evolve an enzymatically active, soluble variant of a hyperthermophilic protein that is normally insoluble when expressed in Escherichia coli, and determining its structure by X-ray crystallography. Analysis of the structure provides insight into the substrate specificity of the enzyme and the improved solubility of the variant.
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