using maltose binding protein as a solubility enhancer and gateway cloning technology
Download
Skip this Video
Download Presentation
Using Maltose-Binding Protein as a Solubility Enhancer AND Gateway™ Cloning Technology

Loading in 2 Seconds...

play fullscreen
1 / 22

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


  • 94 Views
  • Uploaded on

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

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' Using Maltose-Binding Protein as a Solubility Enhancer AND Gateway™ Cloning Technology' - austin-morris


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
using maltose binding protein as a solubility enhancer and gateway cloning technology

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

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

small affinity tags
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

overview of progress to date
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)

advantages of gateway technology
Advantages of Gateway Technology
  • Rapid
  • Efficient
  • Robust
  • Automatable

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

acknowledgements
Acknowledgements

Rachel Kapust*

Karen Routzahn

Jeff Fox*

Matt Bucher*

Joe Tropea

*gone but not forgotten…

ad