Using Maltose-Binding Protein as a Solubility Enhancer AND Gateway

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Using Maltose-Binding Protein as a Solubility
EnhancerANDGateway Cloning Technology
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Advantages of Affinity Tags

• Facilitates Purification Predictable
  Interactions High Specificity Gentle (Indirect)
• Improves Yield Efficient Translation
  Initiation Protection from Proteolysis Enhances
  Solubility

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Systematic Comparison ofSoluble Fusion Partners
Passengers TIMP2, p16, E6, CATD9, GFP, TEV
protease
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Solublility of Fusion Proteins
Kapust Waugh, Protein Science 81668 (1999)
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MBPs from Diverse Archaea and BacteriaAre Potent
Solubility Enhancers
Passengers p16, E6, CATD9, GFP, DHFR,
Rhodanese, Luciferase, G3PDH
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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
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Vector for Controlling Intracellular Processing
of Fusion Proteins
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Delayed Induction of TEV ProteaseImproves the
Solubility of YopN
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Engineering a Functional Multitag
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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

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Summary of Accessory Tag Data
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Putting It All Together
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Structural Proteomics of Type III Secretion in
Yersinia pestis
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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)

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Phage Lambda Recombination in E. coli
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Gateway Cloning Technology
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Piggyback Cloning Strategy
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Anatomy of an MBP Fusion VectorConstructed by
Recombinational Cloning
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Linker Peptides
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Multiple Destination Vectors forRecombinational
Cloning
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Advantages of Gateway Technology

• Rapid
• Efficient
• Robust
• Automatable

but a single entry clone can not be used for
both fused and unfused expression
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Acknowledgements

• Rachel Kapust
• Karen Routzahn
• Jeff Fox
• Matt Bucher
• Joe Tropea

gone but not forgotten