Protein Production at the 100L Scale.

The Baculovirus Expression Vector System (BEVS) has revolutionized the field of recombinant protein expression by enabling efficient and high yield production. The platform offers many advantages including manufacturing speed, flexible design, and scalability. In this chapter, we describe the methods including strategies and considerations to successfully optimize and scale-up using BEVS as a tool for production (Fig.

Peptide-MHC Binding Reveals Conserved Allosteric Sites in MHC Class I- and Class II-Restricted T Cell Receptors (TCRs).

T cells are vital for adaptive immune responses that protect against pathogens and cancers. The T cell receptor (TCR)-CD3 complex comprises a diverse αβ TCR heterodimer in noncovalent association with three invariant CD3 dimers. The TCR is responsible for recognizing antigenic peptides bound to MHC molecules (pMHC), while the CD3 dimers relay activation signals to the T cell.

Antigenicity and Immunogenicity of Differentially Glycosylated Hepatitis C Virus E2 Envelope Proteins Expressed in Mammalian and Insect Cells.

The development of a prophylactic vaccine for hepatitis C virus (HCV) remains a global health challenge. Cumulative evidence supports the importance of antibodies targeting the HCV E2 envelope glycoprotein to facilitate viral clearance. However, a significant challenge for a B cell-based vaccine is focusing the immune response on conserved E2 epitopes capable of eliciting neutralizing antibodies not associated with viral escape.

Peptide-MHC (pMHC) binding to a human antiviral T cell receptor induces long-range allosteric communication between pMHC- and CD3-binding sites.

T cells generate adaptive immune responses mediated by the T cell receptor (TCR)-CD3 complex comprising an αβ TCR heterodimer noncovalently associated with three CD3 dimers. In early T cell activation, αβ TCR engagement by peptide-major histocompatibility complex (pMHC) is first communicated to the CD3 signaling apparatus of the TCR-CD3 complex, but the underlying mechanism is incompletely understood. It is possible that pMHC binding induces allosteric changes in TCR conformation or dynamics that are then relayed to CD3.

Structural Insights into the Inhibitory Mechanism of an Antibody against B7-H6, a Stress-Induced Cellular Ligand for the Natural Killer Cell Receptor NKp30.

Antibodies have been shown to block signaling through cell surface receptors using several mechanisms. The two most common are binding to the ligand-binding site of the receptor and, conversely, binding to the receptor-binding site of the ligand. Here, we investigated the inhibitory mechanism of an antibody (17B1.

Affinity maturation of a broadly neutralizing human monoclonal antibody that prevents acute hepatitis C virus infection in mice.

Unlabelled: Direct-acting antivirals (DAAs) have led to a high cure rate in treated patients with chronic hepatitis C virus (HCV) infection, but this still leaves a large number of treatment failures secondary to the emergence of resistance-associated variants (RAVs). To increase the barrier to resistance, a complementary strategy is to use neutralizing human monoclonal antibodies (HMAbs) to prevent acute infection. However, earlier efforts with the selected antibodies led to RAVs in animal and clinical studies.

Identification of the Docking Site for CD3 on the T Cell Receptor β Chain by Solution NMR.

The T cell receptor (TCR)-CD3 complex is composed of a genetically diverse αβ TCR heterodimer associated noncovalently with the invariant CD3 dimers CD3ϵγ, CD3ϵδ, and CD3ζζ. The TCR mediates peptide-MHC recognition, whereas the CD3 molecules transduce activation signals to the T cell. Although much is known about downstream T cell signaling pathways, the mechanism whereby TCR engagement by peptide-MHC initiates signaling is poorly understood.

T cell receptor bias for MHC: co-evolution or co-receptors?

In contrast to antibodies, which recognize antigens in native form, αβ T cell receptors (TCRs) only recognize antigens as peptide fragments bound to MHC molecules, a feature known as MHC restriction. The mechanism by which MHC restriction is imposed on the TCR repertoire is an unsolved problem that has generated considerable debate. Two principal models have been advanced to explain TCR bias for MHC.

Genetic Association of Peptidoglycan Recognition Protein Variants with Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a common disease, includes Crohn's disease (CD) and ulcerative colitis (UC), and is determined by altered gut bacterial populations and aberrant host immune response. Peptidoglycan recognition proteins (PGLYRP) are innate immunity bactericidal proteins expressed in the intestine. In mice, PGLYRPs modulate bacterial populations in the gut and sensitivity to experimentally induced UC.

Structural changes in the hydrophobic hinge region adversely affect the activity and fidelity of the I260Q mutator DNA polymerase β.

The I260Q variant of DNA polymerase β is an efficient mutator polymerase with fairly indiscriminate misincorporation activities opposite all template bases. Previous modeling studies have suggested that I260Q harbors structural variations in its hinge region. Here, we present the crystal structures of wild type and I260Q rat polymerase β in the presence and absence of substrates.

Hinge residue I174 is critical for proper dNTP selection by DNA polymerase beta.

DNA polymerase beta (pol beta) is the key gap-filling polymerase in base excision repair, the DNA repair pathway responsible for repairing up to 20000 endogenous lesions per cell per day. Pol beta is also widely used as a model polymerase for structure and function studies, and several structural regions have been identified as being critical for the fidelity of the enzyme. One of these regions is the hydrophobic hinge, a network of hydrophobic residues located between the palm and fingers subdomains.