Impact of single-residue mutations on protein thermal stability: The case of threonine 83 of BC2L-CN lectin.

The thermal stability of trimeric lectin BC2L-CN was investigated and found to be considerably altered when mutating residue 83, originally a threonine, located at the fucose-binding loop. Mutants were analyzed using differential scanning calorimetry and isothermal microcalorimetry. Although most mutations decreased the affinity of the protein for oligosaccharide H type 1, six mutations increased the melting temperature (T) by >5 °C; one mutation, T83P, increased the T value by 18.

Engineering a highly durable adeno-associated virus receptor for analytical applications.

Adeno-associated virus (AAV) is a major viral vector used in gene therapy. There are multiple AAV serotypes, and many engineered AAV serotypes are developed to alter their tissue tropisms with capsid modification. The universal AAV receptor (AAVR) is an essential receptor for multiple AAV serotypes.

Biophysical Characterization of the Contribution of the Fab Region to the IgG-FcγRIIIa Interaction.

The cell-surface receptor FcγRIIIa is crucial to the efficacy of therapeutic antibodies as well as the immune response. The interaction of the Fc region of IgG molecules with FcγRIIIa has been characterized, but until recently, it was thought that the Fab regions were not involved in the interaction. To evaluate the influence of the Fab regions in a biophysical context, we carried out surface plasmon resonance analyses using recombinant FcγRIIIa ligands.

Functional expression of the reverse transcriptase αβ heterodimer from avian myeloblastosis virus in Escherichia coli.

The α subunit of avian myeloblastosis virus reverse transcriptase (AMV-RT) is generated from the β-subunit by proteolysis, and the αβ heterodimer represents the active form. The codon-optimized gene was expressed in Escherichia coli, and an active αβ heterodimer was generated. The RNA amplification activity of the purified recombinant AMV-RT αβ heterodimer was similar to that of the native one.

Highly sensitive HPLC analysis and biophysical characterization of N-glycans of IgG-Fc domain in comparison between CHO and 293 cells using FcγRIIIa ligand.

Quality control of monoclonal antibodies is challenging due in part to the diversity of post-translational modifications present. The regulation of the N-glycans of IgG-Fc domain is one of the key factors to maintain the safety and efficacy of antibody drugs. The FcγRIIIa affinity column is an attractive tool for the precise analysis of the N-glycans in IgG-Fc domain.

Assessing the Heterogeneity of the Fc-Glycan of a Therapeutic Antibody Using an engineered FcγReceptor IIIa-Immobilized Column.

The N-glycan moiety of IgG-Fc has a significant impact on multifaceted properties of antibodies such as in their effector function, structure, and stability. Numerous studies have been devoted to understanding its biological effect since the exact composition of the Fc N-glycan modulates the magnitude of effector functions such as the antibody-dependent cell mediated cytotoxicity (ADCC), and the complement-dependent cytotoxicity (CDC). To date, systematic analyses of the properties and influence of glycan variants have been of great interest.

Structural basis for binding of human IgG1 to its high-affinity human receptor FcγRI.

Cell-surface Fcγ receptors mediate innate and adaptive immune responses. Human Fcγ receptor I (hFcγRI) binds IgGs with high affinity and is the only Fcγ receptor that can effectively capture monomeric IgGs. However, the molecular basis of hFcγRI's interaction with Fc has not been determined, limiting our understanding of this major immune receptor.

Engineering of erythropoietin receptor for use as an affinity ligand.

Recombinant human erythropoietin receptor (rhEPOR) has applicability as an affinity ligand for purification of recombinant human erythropoietin (rHuEPO) because of its specific binding to rHuEPO. For application of rhEPOR as a ligand for purification of rHuEPO, soluble rhEPOR was expressed in the periplasm of Escherichia coli and engineered by directed evolution through random mutagenesis and integration of mutations. From the screening of random mutagenesis, we identified an amino acid mutation (H114Y) contributing to rHuEPO binding and four amino acid mutations (R76S, A132D, A162D, and C181Y) contributing to expression of soluble rhEPOR.

Identification of novel non-metal haloperoxidases from the marine metagenome.

Haloperoxidase (HPO, E.C.1.

The binding of soluble recombinant human Fcγ receptor I for human immunoglobulin G is conferred by its first and second extracellular domains.

Human FcγRI is a high affinity receptor for the Fc portion of human immunoglobulin G (IgG), and has extracellular, transmembrane and cytoplasmic regions. The extracellular region of human FcγRI, which is the part that interacts with human IgG, is comprised of three immunoglobulin-like domains. Unlike low affinity Fcγ receptors (FcγRII and FcγRIII), FcγRI has a unique third extracellular domain (D3).

Engineering of recombinant human Fcγ receptor I by directed evolution.

Human FcγRI is a high-affinity receptor for human IgG. On the basis of its binding activity, recombinant human FcγRI (rhFcγRI) has several possible applications, including as a therapeutic reagent to treat immune complex-mediated disease and as a ligand in affinity chromatography for purification of human IgG. As the stability and production rate of rhFcγRI are low, it would need to be engineered for use in such applications.

Cellulomonas soli sp. nov. and Cellulomonas oligotrophica sp. nov., isolated from soil.

Two novel bacterial strains, designated Kc1(T) and Kc5(T), were isolated from soil in Japan. Cells of the novel strains were Gram-reaction-positive, aerobic or facultatively anaerobic, motile rods. Phylogenetic analyses based on 16S rRNA gene sequences indicated that both strains belonged to the genus Cellulomonas.

Effective expression of soluble aglycosylated recombinant human Fcγ receptor I by low translational efficiency in Escherichia coli.

Human FcγRI (CD64) is an integral membrane glycoprotein functioning as a high-affinity receptor binding to monomeric IgG. In this study, the extracellular region of FcγRI, which is the actual part that interacts with IgG, was expressed as aglycosylated recombinant human FcγRI (rhFcγRI) in Escherichia coli. The soluble form of aglycosylated rhFcγRI was expressed in the periplasm of E.

Efficient expression of recombinant soluble human FcγRI in mammalian cells and its characterization.

The extracellular domain of human FcγRI which interacts with a human IgG was expressed as recombinant soluble human FcγRI (rshFcγRI) by Chinese hamster ovary (CHO) cell. Stable CHO cell clones with efficient expression of rshFcγRI were established based on a dihydrofolate reductase (DHFR)/methotrexate (MTX) gene-amplification system. The CHO clones efficiently produced rshFcγRI under high-density continuous culture in a bioreactor.

Efficient production and characterization of recombinant human NELL1 protein in human embryonic kidney 293-F cells.

NELL1 is a secretory protein that induces osteogenic differentiation and bone formation by osteoblastic cells. Because of its potent osteoinductive activity, NELL1 may be useful for bone regeneration therapy. However, at present, we have little knowledge regarding NELL1 receptors and NELL1-mediated signaling pathways.

Fusion protein of interleukin-6 and interleukin-6 receptor without a polypeptide linker.

FP6, a novel recombinant fusion protein of interleukin-6 (IL-6) and IL-6 receptor (IL-6R), was prepared in the methylotrophic yeast Pichia pastoris. This protein was a potent activator of a cell surface transducing glycoprotein, gp130 and is a potential therapeutical reagent in the hemopoietic field. A linker is generally thought to be required for two fused molecules to retain their proper structures although it should preferably be removed to reduce possible antigenicity.

Identification by the phage-display technique of peptides that bind to H7 flagellin of Escherichia coli.

The four peptides interacting with H7 flagellin of Escherichia coli were selected from a phage display library. The library was selected four times, and the interacting phage peptides were competitively eluted with H7 flagellin. An enzyme-linked immunosorbent assay (ELISA) showed that these peptides were reactive with the H7 flagellin in a dose-dependent manner.