Chapter 3. High-throughput protein purification for x-ray crystallography and NMR

Area of Science:

• Structural biology
• Structural genomics
• Protein biochemistry

Background:

• High-quality protein samples are critical for structural biology, particularly for X-ray crystallography and NMR.
• Structural genomics requires high-throughput, cost-effective generation of diverse proteins, often with unknown properties.
• Reproducible purification yielding homogeneous proteins in milligram quantities is a key challenge.

Purpose of the Study:

• To present semiautomated, high-throughput parallel protein purification protocols developed at the Midwest Center for Structural Genomics (MCSG).
• To address the challenges of producing large numbers of diverse, well-characterized proteins for structural genomics.
• To detail purification strategies suitable for generating "structural-biology-grade" proteins.

Main Methods:

• Semiautomated, high-throughput parallel protein purification using fusion proteins with cleavable affinity tags.
• Two-step immobilized metal affinity chromatography (IMAC) with intervening TEV protease tag cleavage.
• Integration of buffer exchange and size exclusion chromatography as needed.
• Implementation on AKTAexplorer and AKTAxpress chromatography workstations.

Main Results:

• Development of robust, reproducible protocols for high-throughput protein purification.
• Successful application of these protocols in structural genomics initiatives, including MCSG and CSGID.
• Generation of milligram quantities of homogeneous proteins suitable for structure determination.

Conclusions:

• Semiautomated, high-throughput purification protocols are effective for producing structural-biology-grade proteins.
• These methods facilitate the goals of structural genomics by increasing protein production efficiency and throughput.
• The described protocols are adaptable and have been integrated into major structural genomics pipelines.