The Skp Chaperone Helps Fold Soluble Proteins <i>in Vitro</i> by Inhibiting Aggregation

The periplasmic seventeen kilodalton protein (Skp) chaperone has been characterized primarily for its role in outer membrane protein (OMP) biogenesis, during which the jellyfish-like trimeric protein encapsulates partially folded OMPs, protecting them from the aqueous environment until delivery to the BAM outer membrane protein insertion complex. However, Skp is increasingly recognized as a chaperone that also assists in folding soluble proteins in the bacterial periplasm. In this capacity, Skp coexpression increases the active yields of many recombinant proteins and bacterial virulence factors. Using a panel of single-chain antibodies and a single-chain T-cell receptor (collectively termed scFvs) possessing varying stabilities and biophysical characteristics, we performed <i>in vivo</i> expression and <i>in vitro</i> folding and aggregation assays in the presence or absence of Skp. For Skp-sensitive scFvs, the presence of Skp during <i>in vitro</i> refolding assays reduced aggregation but did not alter the observed folding rates, resulting in a higher overall yield of active protein. Of the proteins analyzed, Skp sensitivity in all assays correlated with the presence of folding intermediates, as observed with urea denaturation studies. These results are consistent with Skp acting as a holdase, sequestering partially folded intermediates and thereby preventing aggregation. Because not all soluble proteins are sensitive to Skp coexpression, we hypothesize that the presence of a long-lived protein folding intermediate renders a protein sensitive to Skp. Improved understanding of the bacterial periplasmic protein folding machinery may assist in high-level recombinant protein expression and may help identify novel approaches to block bacterial virulence

A New Method to Characterize Conformation-Specific Antibody by a Combination of Agarose Native Gel Electrophoresis and Contact Blotting

In this study, we review the agarose native gel electrophoresis that separates proteins and macromolecular complexes in their native state and transfer of the separated proteins from the agarose gel to membranes by contact blotting which retains the native state of these structures. Green fluorescent protein showed functional state both on agarose gel and blotted membrane. Based on the combined procedures, we discovered conformation-specific monoclonal antibodies against PLXDC2 and SARS-CoV-2 spike protein

Bivalent intra-spike binding provides durability against emergent Omicron lineages: Results from a global consortium

Summary: The SARS-CoV-2 Omicron variant of concern (VoC) and its sublineages contain 31–36 mutations in spike and escape neutralization by most therapeutic antibodies. In a pseudovirus neutralization assay, 66 of the nearly 400 candidate therapeutics in the Coronavirus Immunotherapeutic Consortium (CoVIC) panel neutralize Omicron and multiple Omicron sublineages. Among natural immunoglobulin Gs (IgGs), especially those in the receptor-binding domain (RBD)-2 epitope community, nearly all Omicron neutralizers recognize spike bivalently, with both antigen-binding fragments (Fabs) simultaneously engaging adjacent RBDs on the same spike. Most IgGs that do not neutralize Omicron bind either entirely monovalently or have some (22%–50%) monovalent occupancy. Cleavage of bivalent-binding IgGs to Fabs abolishes neutralization and binding affinity, with disproportionate loss of activity against Omicron pseudovirus and spike. These results suggest that VoC-resistant antibodies overcome mutagenic substitution via avidity. Hence, vaccine strategies targeting future SARS-CoV-2 variants should consider epitope display with spacing and organization identical to trimeric spike