Conversion of scFv peptide-binding specificity for crystal chaperone development

Journal Article · Tue Feb 07 04:00:00 EST 2012 · Protein Eng. Des. Sel.

DOI:https://doi.org/10.1093/protein/gzq120· OSTI ID:1028013

Pai, Jennifer C; Culver, Jeffrey A; Drury, Jason E; Motani, Rakesh S; Lieberman, Raquel L; Maynard, Jennifer A ^[1]

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1. GIT

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 {angstrom} resolution structure of this chaperone reveals a binding surface complementary to the EE peptide and a {approx}52 {angstrom} 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.

• Research Organization: Advanced Photon Source (APS), Argonne National Laboratory (ANL), Argonne, IL (US)
• Sponsoring Organization: NSFOTHERNIH
• OSTI ID: 1028013
• Journal Information: Protein Eng. Des. Sel., Journal Name: Protein Eng. Des. Sel. Journal Issue: (5) ; 01, 2011 Vol. 24; ISSN 1741-0126
• Country of Publication: United States
• Language: ENGLISH

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
AFFINITY
CRYSTAL LATTICES
CRYSTALLIZATION
CRYSTALLOGRAPHY
FILTRATION
MEMBRANE PROTEINS
MUTAGENESIS
PEPTIDES
PROTEIN ENGINEERING
PROTEINS
PURIFICATION
RESOLUTION
SOLUBILITY
SPACE GROUPS
SPECIFICITY