Classical cloning, expression, and purification
Classical cloning, expression, and purification
The ideal protein-expression strategy for X-ray structural analysis should provide correctly folded, soluble, and active protein in sufficient quantities for successful crystallization. Subsequent isolation and purification must be designed to achieve a polished product as rapidly as possible, involving a minimum number of steps. The simplest and least expensive methods employ bacterial hosts such as Escherichia coli, Bacillus, and Staphylococcus but if the target protein is from an eukaryotic source requiring post-translational processing for full functionality, an eukaryotic vector-host system would be appropriate. This chapter discusses the processes of cloning and expression, and protein extraction and isolation.
Keywords: X-ray structural analysis, crystallization, cloning, expression, protein extraction, protein isolation
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