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“universal” purification protocol for a diverse set of proteins

Challenges for HTP protein purification: Q uantity and Q uality. “universal” purification protocol for a diverse set of proteins scale equivalent to cloning and structure determination feasible and economical as few steps as possible as fast as possible

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“universal” purification protocol for a diverse set of proteins

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  1. Challenges for HTP protein purification: Quantity andQuality • “universal” purification protocol for a diverse set of proteins • scale equivalent to cloning and structure determination • feasible and economical • as few steps as possible • as fast as possible • minimize aggregation problem ….. Dementieva et al. 2002

  2. protease time hr t C success rate, % specificity thrombin 16-24 25-37 ~90 -/+ factor Xa 16-60 25-37 ~60 +/- rTEV 1-3 4-30 ~95 + Expression construct selection His thrombin protein pET15b • Affinity tag • small • disorder • easy to remove • (protease) • Protease • specificity • fast • cheap • tagged His thrombin S Xa protein pET30LIC His Xa protein MCSG3 pRroEX(ENLYFQG) His TEV protein MCSG7 (ENLYFQS) His TEV protein Tag cleavage efficiency Dementieva et al. 2002

  3. Purification protocol:as few steps as possible crude • IMAC I usually provides a major • step of the purification • IMAC II removes His-TEV and • persistent contaminant proteins • in E.coli host • Gel-filtration – “polishing” • before crystal optimization IMAC I tag cleavage IMAC II gel filtration Milligram-scale protein production crystallization (screen) 24 20 Crystallization (optimization) Proteinnumber 8 Se-Met protein 2 2 2-5 5-10 10-20 20-40 >50 mg pure protein per 1 L culture Dementieva et al. 2002

  4. With or without tag ? Dementieva et al. 2002

  5. COND UV 7 1 6 8 2 1 7 5 3 2 6 4 3 Pump A PUMP A PUMP B A2 B1 B2 8 1 2 3 5 W2 Affinity + buffer exchange (AKTA platform, Pharmacia) Buffer exchange Semi-automated - chromatography system for simultaneous protein purification MIXER Affinity Buffer exchange Fraction Collector 5 4 Outlet Valve NO NC NO NC 8 1 7 2 6 3 5 4 30 min BufferValve F4 F5 F6 F3 8 8 1 7 1 7 2 6 2 6 3 5 3 5 4 4 11 12 13 14 15 16 17 18 7 Affinity columns Dementieva et al. 2002

  6. Quality control: as fast as possible Dementieva et al. 2002

  7. MCSG Protein Purification Database “Notebook” • Protein Purification Database “Notebook” is fully searchable throughout results of purification. • User is able to use APC number Host identifier or Vector description as keys for his search. • The MCSG Report Field is connected directly to MCSG Report Website (http://www.mcsg.anl.gov/target) and is updating corresponding filed for purification progress report. • SDS Gel Button is connected through the File Report mechanism to Agilent 2100 Bio Sizing Software for separation results. • Chromatography Button invokes connection to File Report mechanism to Pharmacia Purification Robot Software. Dementieva et al. 2002

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