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Using Maltose-Binding Protein as a Solubility Enhancer AND Gateway™ Cloning Technology

Using Maltose-Binding Protein as a Solubility Enhancer AND Gateway™ Cloning Technology. Advantages of Affinity Tags. Facilitates Purification Predictable Interactions High Specificity Gentle (Indirect)

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Using Maltose-Binding Protein as a Solubility Enhancer AND Gateway™ Cloning Technology

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  1. Using Maltose-Binding Protein as a Solubility EnhancerANDGateway™ Cloning Technology

  2. Advantages of Affinity Tags • Facilitates Purification Predictable Interactions High Specificity Gentle (Indirect) • Improves Yield Efficient Translation Initiation Protection from Proteolysis Enhances Solubility

  3. Systematic Comparison ofSoluble Fusion Partners Passengers: TIMP2, p16, E6, CATD9, GFP, TEV protease

  4. Solublility of Fusion Proteins Kapust & Waugh, Protein Science8:1668 (1999)

  5. MBPs from Diverse Archaea and BacteriaAre Potent Solubility Enhancers Passengers: p16, E6, CATD9, GFP, DHFR, Rhodanese, Luciferase, G3PDH

  6. E. coli MBP Can Facilitate the Folding of Its Fusion Partners Active Fusion Proteins CATD9 E6 TEV protease GFP G3PDH DHFR Inactive Fusion Proteins Luciferase Rhodanese

  7. Vector for Controlling Intracellular Processing of Fusion Proteins

  8. Delayed Induction of TEV ProteaseImproves the Solubility of YopN

  9. Engineering a Functional Multitag

  10. Small Affinity Tags Tag Length Sequence Ligand Arg 5 RRRRR cation-exchange resin His 6 HHHHHH Ni-NTA FLAG 8 DYKDDDDK mAb Strep II 8 WSHPQFEK streptavidin BAP 13 LNDIFEAQKIEWH avidin/streptavidin

  11. Summary of Accessory Tag Data

  12. Putting It All Together

  13. Structural Proteomics of Type III Secretion in Yersinia pestis

  14. Overview of Progress to Date Cloned and Expressed 60% (36/60) Soluble Fusion Protein 89% (32/36) Soluble Target Protein 94% (30/32) Purified 67% (16/24) Crystallized 44% (7/16) Structure Solved 71% (5/7)

  15. Phage Lambda Recombination in E. coli

  16. Gateway™ Cloning Technology

  17. Piggyback Cloning Strategy

  18. Anatomy of an MBP Fusion VectorConstructed by Recombinational Cloning

  19. Linker Peptides

  20. Multiple Destination Vectors forRecombinational Cloning

  21. Advantages of Gateway Technology • Rapid • Efficient • Robust • Automatable …but a single entry clone can not be used for both fused and unfused expression

  22. Acknowledgements Rachel Kapust* Karen Routzahn Jeff Fox* Matt Bucher* Joe Tropea *gone but not forgotten…

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