Using maltose binding protein as a solubility enhancer and gateway cloning technology
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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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Presentation Transcript

Advantages of affinity tags
Advantages of Affinity Tags

  • Facilitates Purification Predictable Interactions High Specificity Gentle (Indirect)

  • Improves Yield Efficient Translation Initiation Protection from Proteolysis Enhances Solubility


Systematic comparison of soluble fusion partners
Systematic Comparison ofSoluble Fusion Partners

Passengers: TIMP2, p16, E6, CATD9, GFP, TEV protease


Solublility of fusion proteins
Solublility of Fusion Proteins

Kapust & Waugh, Protein Science8:1668 (1999)


Mbps from diverse archaea and bacteria are potent solubility enhancers
MBPs from Diverse Archaea and BacteriaAre Potent Solubility Enhancers

Passengers: p16, E6, CATD9, GFP, DHFR, Rhodanese, Luciferase, G3PDH


E coli mbp can facilitate the folding of its fusion partners
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



Delayed induction of tev protease improves the solubility of yopn
Delayed Induction of TEV Protease ProteinsImproves the Solubility of YopN



Small affinity tags
Small Affinity Tags Proteins

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





Overview of progress to date
Overview of Progress to Date Proteins

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)





Anatomy of an mbp fusion vector constructed by recombinational cloning
Anatomy of an MBP Fusion Vector ProteinsConstructed by Recombinational Cloning


Linker peptides
Linker Peptides Proteins


Multiple destination vectors for recombinational cloning
Multiple Destination Vectors for ProteinsRecombinational Cloning


Advantages of gateway technology
Advantages of Gateway Technology Proteins

  • Rapid

  • Efficient

  • Robust

  • Automatable

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


Acknowledgements
Acknowledgements Proteins

Rachel Kapust*

Karen Routzahn

Jeff Fox*

Matt Bucher*

Joe Tropea

*gone but not forgotten…


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