Production of F(ab')2 from Monoclonal and Polyclonal Antibodies.

Antibodies are widely used in therapeutic, diagnostic, and research applications, and antibody derivatives such as F(ab')2 fragments are used when only a particular antibody region is required. F(ab')2 can be produced through antibody engineering, but some applications require F(ab')2 produced from an original formulated antibody or directly from a polyclonal antibody pool. The cysteine protease immunoglobulin-degrading enzyme (IdeS) from Streptococcus pyogenes digests immunoglobulin G (IgG) specifically and efficiently to produce F(ab')2 . Here we detail the production and purification of recombinant IdeS; its utilization to digest monoclonal or polyclonal antibodies to F(ab')2 fragments; and F(ab')2 purification through consecutive affinity chromatography steps. The resultant F(ab')2 exhibit high purity, retain antigen-binding functionality, and are readily utilizable in various downstream applications. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Production and purification of F(ab')2 fragments from monoclonal and polyclonal antibodies using IdeS Alternate Protocol: Purification of polyclonal antigen-specific F(ab')2 fragments from human serum or secretions Support Protocol: Production and purification of IdeS.

The Lys-Specific Molecular Tweezer, CLR01, Modulates Aggregation of the Mutant p53 DNA Binding Domain and Inhibits Its Toxicity.

The tumor suppressor p53 plays a unique role as a central hub of numerous cell proliferation and apoptotic pathways, and its malfunction due to mutations is a major cause of various malignancies. Therefore, it serves as an attractive target for developing novel anticancer therapeutics. Because of its intrinsically unstable DNA binding domain, p53 unfolds rapidly at physiological temperature. Certain mutants shift the equilibrium toward the unfolded state and yield high-molecular weight, nonfunctional, and cytotoxic ß-sheet-rich aggregates that share tinctorial and conformational similarities with amyloid deposits found in various protein misfolding diseases. Here, we examined the effect of a novel protein assembly modulator, the lysine (Lys)-specific molecular tweezer, CLR01, on different aggregation stages of misfolded mutant p53 in vitro and on the cytotoxicity of the resulting p53 aggregates in cell culture. We found that CLR01 induced rapid formation of ß-sheet-rich, intermediate-size p53 aggregates yet inhibited further p53 aggregation and reduced the cytotoxicity of the resulting aggregates. Our data suggest that aggregation modulators, such as CLR01, could prevent the formation of toxic p53 aggregates.