Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization

Johnson, J.L.; Entzminger, K.C.; Hyun, J.; Kalyoncu, S.; Heaner, D.P.; Morales, I.A.; Sheppard, A.; Gumbart, J.C.; Maynard, J.A.; Lieberman, R.L.

doi:10.1107/S1399004715001856

Acta Crystallographica Section D
Acta Crystallographica
Section D STRUCTURAL BIOLOGY

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Figure 3
Fab/EE binding to EE-tagged membrane proteins. (a) ELISA analysis of Fab/EE binding to A₂aR-GFP. Purified A₂aR-GFP proteins with or without the EE-peptide insertion were added to ELISA wells coated with Fab/EE or blocked control wells. Captured protein was detected using an anti-GFP antibody. (b, c) SPR analysis of binding. GPCRs were injected in duplicate for each concentration tested and binding to immobilized Fab/EE was monitored. Average traces are shown and demonstrate concentration-dependent binding to A₂aR-GFP-EE. No binding was observed for A₂aR-GFP lacking the EE peptide. (d) Elution profile of purified Fab/EE incubated with WT intimin, intimin-EE1 or intimin-EE2. Reducing SDS–PAGE analysis (inset) of fractions selected from 10.5 to 13 ml elution volumes shows coelution of Fab/EE–intimin-EE1 and Fab/EE–intimin-EE2.

STRUCTURAL
BIOLOGY

ISSN: 2059-7983

Volume 71| Part 4| April 2015| Pages 896-906

https://doi.org/10.1107/S1399004715001856

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