Efficient and economic protein labeling for NMR in mammalian expression systems: Application to a preT-cell and T-cell receptor protein.

Protein nuclear magnetic resonance (NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with N and C, respectively. Moreover, E.

Thermostability of a recombinant G protein-coupled receptor expressed at high level in mammalian cell culture.

Rational design of pharmaceutical drugs targeting integral membrane G protein-coupled receptors (GPCR) requires thorough understanding of ligand binding and mechanism of activation through high resolution structural studies of purified proteins. Due to inherent conformational flexibility of GPCR, stabilization of these proteins solubilized from cell membranes into detergents is a challenging task. Here, we take advantage of naturally occurring post-translational modifications for stabilization of purified GPCR in detergent micelles.

Glycan characterization of pregnancy-specific glycoprotein 1 and its identification as a novel Galectin-1 ligand.

Pregnancy-specific beta 1 glycoprotein (PSG1) is secreted from trophoblast cells of the human placenta in increasing concentrations as pregnancy progresses, becoming one of the most abundant proteins in maternal serum in the third trimester. PSG1 has seven potential N-linked glycosylation sites across its four domains. We carried out glycomic and glycoproteomic studies to characterize the glycan composition of PSG1 purified from serum of pregnant women and identified the presence of complex N-glycans containing poly LacNAc epitopes with α2,3 sialyation at four sites.

A high density CHO-S transient transfection system: Comparison of ExpiCHO and Expi293.

Chinese Hamster Ovary (CHO) cells are the principal mammalian host used for stable cell line generation and biotherapeutic protein production. Until recently, production of milligrams to grams of protein in CHO transient systems was challenging. As such, Human Embryonic Kidney (HEK293) cells are the most common mammalian cell type used for transient transfection.

Scalable transient protein expression.

Transient transfection is a well-established method to rapidly express recombinant proteins from mammalian cells. Accelerating activity in biotherapeutic drug development, demand for protein-based reagents, vaccine research, and large initiatives in structural and functional studies of proteins have propelled the need to generate moderate to high amounts of recombinant proteins and other macromolecules in a flexible and rapid manner. Progress over the last 10-15 years has demonstrated that transient transfections can be reliably and readily scaled up to handle milliliters to tens of liters of cells in suspension culture and obtain milligrams to grams of recombinant protein in a process that requires only days to weeks.

A phase 1 study of PAmAb, a fully human monoclonal antibody against Bacillus anthracis protective antigen, in healthy volunteers.

Background: Inhibition of the binding of Bacillus anthracis protective antigen (PA) to its cellular receptor can abrogate the downstream toxin-mediated deleterious effects of the anthrax toxin. A fully human monoclonal antibody against B. anthracis PA, PAmAb, was previously shown to provide a survival advantage in rabbit and monkey models of inhalational anthrax.

Scalable purification of Bacillus anthracis protective antigen from Escherichia coli.

The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor that are produced by the Gram-positive bacterium, Bacillus anthracis. Current vaccines against anthrax use PA as their primary component. In this study, we developed a scalable process to produce and purify multi-gram quantities of highly pure, recombinant PA (rPA) from Escherichia coli.

Development of an edema factor-mediated cAMP-induction bioassay for detecting antibody-mediated neutralization of anthrax protective antigen.

Intoxication of mammalian cells by Bacillus anthracis requires the coordinate activity of three distinct bacterial proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). Among these proteins, PA has become the major focus of work on monoclonal antibodies and vaccines designed to treat or prevent anthrax infection since neither EF nor LF is capable of inducing cellular toxicity in its absence. Here, we present the development of a sensitive, precise, and biologically relevant bioassay platform capable of quantifying antibody-mediated PA neutralization.