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Protein Eng Des Sel. 2011 May;24(5):419-28. doi: 10.1093/protein/gzq120. Epub 2011 Jan 8.

Conversion of scFv peptide-binding specificity for crystal chaperone development.

Author information

1
Department of Chemical Engineering, University of Texas at Austin, TX 78712, USA.

Abstract

In spite of advances in protein expression and purification over the last decade, many proteins remain recalcitrant to structure determination by X-ray crystallography. One emerging tactic to obtain high-quality protein crystals for structure determination, particularly in the case of membrane proteins, involves co-crystallization with a protein-specific antibody fragment. Here, we report the development of new recombinant single-chain antibody fragments (scFv) capable of binding a specific epitope that can be introduced into internal loops of client proteins. The previously crystallized hexa-histidine-specific 3D5 scFv antibody was modified in the complementary determining region and by random mutagenesis, in conjunction with phage display, to yield scFvs with new biochemical characteristics and binding specificity. Selected variants include those specific for the hexa-histidine peptide with increased expression, solubility (up to 16.6 mg/ml) and sub-micromolar affinity, and those with new specificity for the EE hexa-peptide (EYMPME) and nanomolar affinity. Complexes of one such chaperone with model proteins harboring either an internal or a terminal EE tag were isolated by gel filtration. The 3.1 Å resolution structure of this chaperone reveals a binding surface complementary to the EE peptide and a ∼52 Å channel in the crystal lattice. Notably, in spite of 85% sequence identity, and nearly identical crystallization conditions, the engineered scFv crystallizes in a different space group than the parent 3D5 scFv, and utilizes two new crystal contacts. These engineered scFvs represent a new class of chaperones that may eliminate the need for de novo identification of candidate chaperones from large antibody libraries.

PMID:
21217145
PMCID:
PMC3077810
DOI:
10.1093/protein/gzq120
[Indexed for MEDLINE]
Free PMC Article

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