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From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins
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JoVE Journal Chemistry
From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins

From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins

DOI:
10.3791/53245-v

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09:55 min

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July 04, 2016

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, , , ,
1Department of Biological Sciences, Markey Center for Structural Biology,Purdue University, 2National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK),National Institutes of Health, 3National Institute of General Medical Sciences (NIGMS),National Institutes of Health

Chapters

  • 00:05Title
  • 00:46In Vivo Membrane-targeted Expression
  • 03:09Isolation from Membrane Fractions
  • 04:41Purification Using Immobilized Metal-affinity Chromatography (IMAC), Ion-exchange, and Gel Filtration
  • 07:09Heat Modifiability Assay
  • 08:35Results: Crystals to Structure Determination for the Putative Vaccine Target YiuR from Yersinia pestis
  • 09:29Conclusion

Summary

Automatic Translation

Automatic Translation

β-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of β-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

Tags

Keywords: Outer Membrane ProteinsBeta-barrel ProteinsMembrane Protein FoldingProtein ExpressionProtein PurificationProtein CrystallizationT7 Expression VectorBL21(DE3) CellsIPTG InductionSDS-PAGE

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