This chapter presents in detail the process used in high throughput bacterial production of recombinant human proteins for crystal structure determination. The core principles are: (1) Generating at least 10 truncated constructs from each target gene. (2) Ligation-independent cloning (LIC) into a bacterial expression vector. All proteins are expressed with an N-terminal, TEV protease cleavable fusion peptide. (3) Small-scale test expression to identify constructs producing soluble protein. (4) Liter-scale production in shaker flasks. (5) Purification by Ni-affinity chromatography and gel filtration. (6) Protein characterization and preparation for crystallography. The chapter also briefly presents alternative procedures, to be applied based on specific knowledge of protein families or when the core protocol is unsatisfactory. This scheme has been applied to more than 550 human proteins (>10,000 constructs) and has resulted in the deposition of 112 unique structures. The methods presented do not depend on specialized equipment or robotics; hence, they provide an effective approach for handling individual proteins in a regular research lab.

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