Solubilization and Humanization of Paraoxonase-1

Paraoxonase-1 (PON1) is a serum protein, the activity of which is related to susceptibility to cardiovascular disease and intoxication by organophosphorus (OP) compounds. It may also be involved in innate immunity, and it is a possible lead molecule in the development of a catalytic bioscavenger of OP pesticides and nerve agents. Human PON1 expressed in E. coli is mostly found in the insoluble fraction, which motivated the engineering of soluble variants, such as G2E6, with more than 50 mutations from huPON1. We examined the effect on the solubility, activity, and stability of three sets of mutations designed to solubilize huPON1 with fewer overall changes: deletion of the N-terminal leader, polar mutations in the putative HDL binding site, and selection of the subset of residues that became more polar in going from huPON1 to G2E6. All three sets of mutations increase the solubility of huPON1; the HDL-binding mutant has the largest effect on solubility, but it also decreases the activity and stability the most. Based on the G2E6 polar mutations, we “humanized” an engineered variant of PON1 with high activity against cyclosarin (GF) and found that it was still very active against GF with much greater similarity to the human sequence

Dextran as a Generally Applicable Multivalent Scaffold for Improving Immunoglobulin-Binding Affinities of Peptide and Peptidomimetic Ligands

Molecules able to bind the antigen-binding sites of antibodies are of interest in medicine and immunology. Since most antibodies are bivalent, higher affinity recognition can be achieved through avidity effects in which a construct containing two or more copies of the ligand engages both arms of the immunoglobulin simultaneously. This can be achieved routinely by immobilizing antibody ligands at high density on solid surfaces, such as ELISA plates, but there is surprisingly little literature on scaffolds that routinely support bivalent binding of antibody ligands in solution, particularly for the important case of human IgG antibodies. Here we show that the simple strategy of linking two antigens with a polyethylene glycol (PEG) spacer long enough to span the two arms of an antibody results in higher affinity binding in some, but not all, cases. However, we found that the creation of multimeric constructs in which several antibody ligands are displayed on a dextran polymer reliably provides much higher affinity binding than is observed with the monomer in all cases tested. Since these dextran conjugates are simple to construct, they provide a general and convenient strategy to transform modest affinity antibody ligands into high affinity probes. An additional advantage is that the antibody ligands occupy only a small number of the reactive sites on the dextran, so that molecular cargo can be attached easily, creating molecules capable of delivering this cargo to cells displaying antigen-specific receptors

Site-Specific Dual Antibody Conjugation via Engineered Cysteine and Selenocysteine Residues

Site-specific conjugation technologies enable the production of homogeneous antibody–drug conjugates (ADCs) with improved therapeutic indices compared to conventional ADCs. However, current site-specific conjugation methods can only attach one type of drug to a single antibody. Given the emergence of acquired resistance to current ADCs, arming single antibodies with different drugs may provide an attractive option in the development of next-generation ADCs. Here, we describe a site-specific dual conjugation strategy as a platform for dual warhead ADCs

Clinical and pathological features of <i>BRCA1</i> c.190T>C related ovarian cancer cases.

<p>Clinical and pathological features of <i>BRCA1</i> c.190T>C related ovarian cancer cases.</p

Clinical and pathological features of <i>BRCA1</i> c.190T>C related breast cancer cases.

a<p>Ductal and lobular type.</p

Semi-quantitative fragment analysis of the Δexon5q isoform.

<p>The upper panel shows the capillary electrophoresis patterns of the cDNA fragments spanning <i>BRCA1</i> exons 5 and 6 observed in LCLs from a <i>BRCA1</i> wild type individual, and from carriers of the c.190T>C and c.212G>A, which causes the up-regulation of the Δexon5q transcript, mutations. The Δexon5q and full-length (FL) isoforms are indicated. The lower panel shows the ratio between the peak areas of the Δexon5q and full-length isoforms. The LCLs were cultured in the presence (dark grey bars) and in the absence (light grey bar) of cycloheximide. Control bars represent the average value observed in six wild-type LCLs. c.190T>C bars represent the average value observed in four mutant LCLs. The error bars represent standard deviation.</p

Haplotype branching trees in families segregating the <i>BRCA1</i> c.190T>C (p.Cys64Arg).

<p>The six short tandem repeat markers analyzed are shown together with their position in the Marshfield genetic map. Family haplotypes are indicated with the corresponding family ID codes. The most common haplotype is indicated in bold numbers.</p