Protein production from HEK293 cell line-derived stable pools with high protein quality and quantity to support discovery research.

Antibody-based therapeutics and recombinant protein reagents are often produced in mammalian expression systems, which provide human-like post-translational modifications. Among the available mammalian cell lines used for recombinant protein expression, Chinese hamster ovary (CHO)-derived suspension cells are generally utilized because they are easy to culture and tend to produce proteins in high yield. However, some proteins purified from CHO cell overexpression suffer from clipping and display undesired non-human post translational modifications (PTMs).

Immunotherapy combinations overcome resistance to bispecific T cell engager treatment in T cell-cold solid tumors.

Therapeutic approaches are needed to promote T cell-mediated destruction of poorly immunogenic, "cold" tumors typically associated with minimal response to immune checkpoint blockade (ICB) therapy. Bispecific T cell engager (BiTE) molecules induce redirected lysis of cancer cells by polyclonal T cells and have demonstrated promising clinical activity against solid tumors in some patients. However, little is understood about the key factors that govern clinical responses to these therapies.

Automated buffer preparation using quaternary valve in fast performance liquid chromatography for protein purification from a cell membrane.

There is a great need for high-throughput protein purification to produce protein molecules for research and therapeutics. Although there have been significant advancements made in automated multi-step chromatography and preparative in-process design-of-experiment (DOE) capabilities in commercial fast performance liquid chromatography (FPLC) instruments, almost all commercial FPLCs rely on a binary buffer mixing system, which hinders automated buffer preparation. Nevertheless, current-generation FPLCs are equipped with a quaternary mixer designed for limited in-line buffer preparation and preparative pH scouting DOE experiments.

Membrane cholesterol modulates STEAP2 conformation during dynamic intracellular trafficking processes leading to broad subcellular distribution.

STEAP2 is a member of the Six-Transmembrane Epithelial Antigen of the Prostate (STEAP) protein family that is proposed to function as metalloreductase. While STEAP2 shows a complex subcellular distribution pattern localizing to both secretory and endocytic pathway organelles, how such broad steady-state distribution is maintained is unknown. Similarly, whether STEAP2 undergoes any compartment-specific modulation during intracellular trafficking has not been reported.

Novel protein therapeutic joint retention strategy based on collagen-binding Avimers.

Designing drugs to treat diseases associated with articular joints, particularly those targeting chondrocytes, is challenging due to unique local environmental constraints including the avascular nature of cartilage, the absence of a closed joint compartment, and a highly cross-linked extracellular matrix. In an effort to address these challenges, we developed a novel strategy to prolong residence time of intra-articularly administered protein therapeutics. Avimer domains are naturally found in membrane polypeptides and mediate diverse protein-protein interactions.

Auto-induction of Pichia pastoris AOX1 promoter for membrane protein expression.

Pichia pastoris is a highly successful recombinant protein expression system due to its ability to quickly generate large quantities of recombinant proteins in simple media. P. pastoris has been used to successfully generate milligram quantities of many important human membrane proteins, including G-protein coupled receptors, ion channels, and transporters, which are becoming increasingly important therapeutic targets.

FGF21 can be mimicked in vitro and in vivo by a novel anti-FGFR1c/β-Klotho bispecific protein.

The endocrine hormone FGF21 has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. As an alternative to the native cytokine, we generated bispecific Avimer polypeptides that bind with high affinity and specificity to one of the receptor and coreceptor pairs used by FGF21, FGFR1c and β-Klotho. These Avimers exhibit FGF21-like activity in in vitro assays with potency greater than FGF21.

Signal integration by DegS and RseB governs the σ E-mediated envelope stress response in Escherichia coli.

In Escherichia coli, the σ(E) transcription factor monitors and maintains outer membrane (OM) integrity by activating genes required for assembly of its two key components, outer membrane proteins (OMPs) and lipopolysaccharide (LPS) and by transcribing small RNAs to down-regulate excess unassembled OMPs. σ(E) activity is governed by the rate of degradation of its membrane-spanning anti-σ factor, RseA. Importantly, the DegS protease can initiate RseA cleavage only when activated by binding to unassembled OMPs.

Fine-tuning of the Escherichia coli sigmaE envelope stress response relies on multiple mechanisms to inhibit signal-independent proteolysis of the transmembrane anti-sigma factor, RseA.

Proteolytic cascades are widely implicated in signaling between cellular compartments. In Escherichia coli, accumulation of unassembled outer membrane porins (OMPs) in the envelope leads to expression of sigma(E)-dependent genes in the cytoplasmic cellular compartment. A proteolytic cascade conveys the OMP signal by regulated proteolysis of RseA, a membrane-spanning anti-sigma factor whose cytoplasmic domain inhibits sigma(E)-dependent transcription.

Regulation of the Escherichia coli sigma-dependent envelope stress response.

The Escherichia colisigma(E)-dependent stress response pathway controls the expression of genes encoding periplasmic folding catalysts, proteases, biosynthesis enzymes for lipid A (a component of lipopolysaccharide or LPS) and other proteins known or predicted to function in or produce components of the envelope. When E. coli is subjected to heat or other stresses that generate unfolded envelope proteins, sigma(E) activity is induced.

OMP peptide signals initiate the envelope-stress response by activating DegS protease via relief of inhibition mediated by its PDZ domain.

Transmembrane signaling between intracellular compartments is often controlled by regulated proteolysis. Escherichia coli respond to misfolded or unfolded outer-membrane porins (OMPs) in the periplasm by inducing sigma(E)-dependent transcription of stress genes in the cytoplasm. This process requires a proteolytic cascade initiated by the DegS protease, which destroys a transmembrane protein (RseA) that normally binds to and inhibits sigma(E).

DegS and YaeL participate sequentially in the cleavage of RseA to activate the sigma(E)-dependent extracytoplasmic stress response.

All cells have stress response pathways that maintain homeostasis in each cellular compartment. In the Gram-negative bacterium Escherichia coli, the sigma(E) pathway responds to protein misfolding in the envelope. The stress signal is transduced across the inner membrane to the cytoplasm via the inner membrane protein RseA, the anti-sigma factor that inhibits the transcriptional activity of sigma(E).