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Protein synthesis and secretion are essential to cellular life. Although secretory activities may vary in different cell types, what determines the maximum secretory capacity is inherently difficult to study. Increasing protein synthesis until reaching the limit of secretory capacity is one strategy to address this key issue. Under highly optimized growth conditions, recombinant CHO cells engineered to produce a model human IgG clone started housing rod-shaped crystals in the endoplasmic reticulum (ER) lumen. The intra-ER crystal growth was accompanied by cell enlargement and multinucleation and continued until crystals outgrew cell size to breach membrane integrity. The intra-ER crystals were composed of correctly folded, endoglycosidase H-sensitive IgG. Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG, and the crystallization was reproduced in vitro by exposing a high concentration of IgG to a near neutral pH. The striking cellular phenotype implicated the efficiency of IgG protein synthesis and oxidative folding exceeded the capacity of ER export machinery. As a result, export-ready IgG accumulated progressively in the ER lumen until a threshold concentration was reached to nucleate crystals. Using an in vivo system that reports accumulation of correctly folded IgG, we showed that the ER-toGolgi transport steps became rate-limiting in cells with high secretory activity.Immunoglobulins continue to serve as an important model secretory cargo for investigating biochemical processes of oxidative protein folding and subunit assembly in the ER 2 lumen (1). Although immunoglobulins are indispensable as research tools, their potential as human therapeutics has attracted significant interest in recent years in the manufacture of human IgG at large scale (2, 3). Therapeutic human IgGs are often recombinantly produced in variants of CHO cells that were adapted to propagate in suspension culture format. Mammalian cell hosts are often preferred for biopharmaceutical production not only just to achieve desired co-translational and post-translational modifications (4) but also to exploit the stringent protein quality control mechanisms that only allow the secretion of properly folded and correctly assembled proteins (5, 6).To achieve high recombinant protein expression levels in mammalian cells, various cis-acting exogenous nucleotide elements have been engineered into transgene expression cassettes to enhance transcription efficiency, extend message halflife, and increase translation initiation frequency (7,8). Exogenous nucleotide elements also enabled strategies to increase transgene copy number by gene amplification and to suppress epigenetic silencing (9 -12). Despite the success in boosting protein expression per se through these expression vector engineering approaches, such enhancements did not translate into higher glycoprotein secretion partly because post-translational events such as protein folding/assembly and intracellular vesicular transport steps along the secreto...

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Antibody solutions are typically much more viscous than solutions of globular proteins at equivalent volume fraction. Here we propose that this is due to molecular entanglements that are caused by the elongated shape and intrinsic flexibility of antibody molecules. We present a simple theory in which the antibodies are modeled as linear polymers that can grow via reversible bonds between the antigen binding domains. This mechanism explains the observation that relatively subtle changes to the interparticle interaction can lead to large changes in the viscosity. The theory explains the presence of distinct power law regimes in the concentration dependence of the viscosity as well as the correlation between the viscosity and the charge on the variable domain in our anti-streptavidin IgG 1 model system.

Phosphoinositides play important roles in numerous intracellular membrane pathways. Little is known about the regulation or function of these lipids in rod photoreceptor cells, which have highly active membrane dynamics. Using new assays with femtomole sensitivity, we determined that whereas levels of phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate were below detection limits, phosphatidylinositol-3-phosphate (PI(3)P) levels in rod inner/outer segments increased more than 30-fold after light exposure. This increase was blocked in a rod-specific knockout of the PI-3 kinase Vps34, resulting in failure of endosomal and autophagy-related membranes to fuse with lysosomes, and accumulation of abnormal membrane structures. At early ages, rods displayed normal morphology, rhodopsin trafficking, and light responses, but underwent progressive neurodegeneration with eventual loss of both rods and cones by twelve weeks. The degeneration is considerably faster than in rod knockouts of autophagy genes, indicating defects in endosome recycling or other PI(3)P-dependent membrane trafficking pathways are also essential for rod survival.