Antibody aggregation is frequently mediated by the complementarity determining regions within the variable domains and can significantly decrease purification yields, shorten shelf-life and increase the risk of anti-drug immune responses. Aggregation-resistant antibodies could offset these risks; accordingly, we have developed a directed evolution strategy to improve Fab stability. A Fab-phage display vector was constructed and the VH domain targeted for mutagenesis by error-prone PCR. To enrich for thermoresistant clones, the resulting phage library was transiently heated, followed by selection for binding to an anti-light chain constant domain antibody. Five unique variants were identified, each possessing one to three amino acid substitutions. Each engineered Fab possessed higher, Escherichia coli expression yield, a 2-3°C increase in apparent melting temperature and improved aggregation resistance upon heating at high concentration. Select mutations were combined and shown to confer additive improvements to these biophysical characteristics. Finally, the wild-type and most stable triple variant Fab variant were converted into a human IgG1 and expressed in mammalian cells. Both expression level and aggregation resistance were similarly improved in the engineered IgG1. Analysis of the wild-type Fab crystal structure provided a structural rationale for the selected residues changes. This approach can help guide future Fab stabilization efforts.
Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.
Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although non-complementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts.
MICAL (Molecule Interacting with CasL) is a 1048 amino acid protein consisting of a monooxygenase domain (FD) with redox activity, a Calponin homology domain (CH), a LIM domain, a proline-rich region, and a C-term region containing coiled-coil ERM a-like domain. In axon guidance, MICAL is a key molecule that links the extracellular signal from semaphorins -a class of repulsive guidance cues-to the reorganization of the cytoskeleton. Proper axon guidance, the process by which growing axons respond to extracellular cues that guide them towards their appropriate targets, is vital in neural development processes such as neuronal cell-migration, axonal branching, path finding, and fasiculation/defasiculation. Our laboratory has previously determined the crystal structure of MICALs FD domain (MICAL FD ) and showed that it uses NADPH as the reductant. Studies showed that MICAL FD and MICAL FD-CH can bind and oxidize Met44 on actin filaments, thereby affecting their polymerization dynamics. However, modulation of these MICAL activities by its non-redox domains is poorly understood. To structurally characterize the modulation by the CH domain, we determined the crystal structure of MICAL FD-CH to 3.0-Å resolution. The structure reveals that the CH domain does not interact with the active site in the FD domain. Furthermore, the FD and CH domains are flexible with respect to each other; MICAL FD-CH crystallized in two different crystal forms, and no electron density was observed for the 18-residue linker between the two domains. In actin-binding proteins with tandem CH domains, the flexibility of the domains with respect to each other is important for binding F-actin. Similarly, the flexibility of the two domains in MICAL FD-CH may be important in optimizing the binding to F-actin such that Met44 is more accessible to the active site.
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