Growth/differentiation factor 15 (GDF15), also known as MIC-1, is a distant member of the transforming growth factor-β (TGF-β) superfamily and has been implicated in various biological functions, including cancer cachexia, renal and heart failure, atherosclerosis and metabolism. A connection between GDF15 and body-weight regulation was initially suggested on the basis of an observation that increasing GDF15 levels in serum correlated with weight loss in individuals with advanced prostate cancer. In animal models, overexpression of GDF15 leads to a lean phenotype, hypophagia and other improvements in metabolic parameters, suggesting that recombinant GDF15 protein could potentially be used in the treatment of obesity and type 2 diabetes. However, the signaling and mechanism of action of GDF15 are poorly understood owing to the absence of a clearly identified cognate receptor. Here we report that GDNF-family receptor α-like (GFRAL), an orphan member of the GFR-α family, is a high-affinity receptor for GDF15. GFRAL binds to GDF15 in vitro and is required for the metabolic actions of GDF15 with respect to body weight and food intake in vivo in mice. Gfral mice were refractory to the effects of recombinant human GDF15 on body-weight, food-intake and glucose parameters. Blocking the interaction between GDF15 and GFRAL with a monoclonal antibody prevented the metabolic effects of GDF15 in rats. Gfral mRNA is highly expressed in the area postrema of mouse, rat and monkey, in accordance with previous reports implicating this region of the brain in the metabolic actions of GDF15 (refs. 4,5,6). Together, our data demonstrate that GFRAL is a receptor for GDF15 that mediates the metabolic effects of GDF15.

Protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. We describe a protein purification scheme wherein Ralstonia eutropha is used to produce its own "affinity matrix," thereby eliminating the need for external chromatographic purification steps. This approach is based on the specific interaction of phasin proteins with granules of the intracellular polymer polyhydroxybutyrate (PHB). By creating in-frame fusions of phasins and green fluorescent protein (GFP) as a model protein, we demonstrated that GFP can be efficiently sequestered to the surface of PHB granules. In a second step, we generated a phasin-intein-GFP fusion, wherein the self-cleaving intein can be activated by the addition of thiols. This construct allowed for the controlled binding and release of essentially pure GFP in a single separation step. Finally, pure, active ␤-galactosidase was obtained in a single step using the above described method.We have previously reported the development of a novel high cell density protein expression platform based on the gram-negative bacterium Ralstonia eutropha (22,23). This system has been developed to overcome some of the shortcomings associated with recombinant protein expression in Escherichia coli (e.g., poor fermentation performance at high cell density, and inclusion body formation). Expression of organophosphohydrolase, an enzyme originally isolated from Pseudomonas diminuta (20) and prone to inclusion body formation in Escherichia coli (4,28,29), was demonstrated at high levels. Titers of active, soluble organophosphohydrolyase, in excess of 10 g/liter were obtained in high cell density fermentation (3), representing at least a 100-fold increase over those previously reported in E. coli.While the successful expression of a recombinant protein is a necessary requirement, recovery and purification still remain a significant cost in recombinant protein production. We thus sought to integrate the existing R. eutropha protein expression platform with a protein purification strategy to simplify the expression and purification of recombinant proteins. This specific approach uses the natural ability of R. eutropha to produce a polymer known as polyhydroxybutyrate (PHB), which accumulates as insoluble granules within the cell. PHB is a member of the polyhydroxyalkanoate class of polymers, synthesized by many bacteria, as carbon storage compounds (2,15,16,26,30,31,32). Polyhydroxyalkanoates have received attention as biodegradable polymers and can be obtained by fermentation processes utilizing cheap, abundant renewable carbon sources (2, 24). Polyhydroxyalkanoates have been produced industrially by ZENECA Bioproducts (26) and Monsanto (10).PHB synthesis in R. eutropha has been the model system for studying polyhydroxyalkanoate biosynthesis in bacteria (10,15,16,18,26). The biogenesis of polyhydroxyalkanoate granules involves two distinct proteins, the polyhydroxyalkanoate synthase...

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS-KO host cell line. By creating a bi-allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS-KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)-mediated transfections were performed at high cell density (4 × 10(6) cells/mL) followed by immediate growth arrest at 32 °C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre-complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7-day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc-fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7-day process. Titers were increased to 1 g/L by extending the culture to 16 days. We also present two case studies comparing product quality of material generated by transient HEK293, transient CHO K1SV GS-KO, and stable CHO K1SV KO pool. Protein from transient CHO was more representative of stable CHO protein compared to protein produced from HEK293.

We describe the development of a novel protein expression system based on the industrial fermentation organism Ralstonia eutropha (formerly known as Alcaligenes eutrophus) NCIMB 40124. This new system overcomes some of the shortcomings of traditional Escherichia coli-based protein expression systems, particularly the propensity of such systems to form inclusion bodies during high-level expression. Using a proteomics approach, we identified promoters that can be induced by simple process parameters or medium compositions in high-density cell culture or shake flasks, respectively. By combining newly developed molecular biological tools with a high-cell-density fermentation process, we were able to produce high levels (>1 g/liter) of soluble, active organophosphohydrolase, a model enzyme prone to inclusion body formation in E. coli.Advances in protein engineering, the completion of numerous bacterial and fungal genome sequencing projects, and the isolation of new genes from extremophiles have led to an increased number of useful proteins. However, to enable recombinant proteins to play a role in applications where larger quantities are required, such as tissue engineering or catalytic materials, production technologies that are more efficient and robust are desirable. Improving protein production is the primary goal of recombinant microbial process development and is a focus of our laboratory. Overall protein productivity can be improved by increasing the product of two variables: (i) the amount of recombinant protein per cell (specific productivity) and (ii) the amount of cell mass per unit of volume and time (cell productivity). In order to improve volumetric productivity in a cost-effective manner, recombinant proteins are often produced in high-cell-density fermentations. High-cell-density fermentations offer many advantages over traditional fermentations in that final product concentrations are higher and downtime and water usage are reduced, yet overall productivity is improved, resulting in lower setup and operating costs (3, 6).In the absence of specific folding or posttranslational modification requirements, Escherichia coli is usually the expression host of choice. E. coli-based fermentation systems produce good yields at laboratory scale; however, scale up to industrial scale, where oxygen enrichment, dialysis, and sophisticated feeding algorithms become impractical and cost prohibitive, has been challenging (22). Unlike laboratory-scale fermentors, where the above-mentioned techniques are feasible, largescale industrial fermentors are limited by mixing constraints and their ability to transfer oxygen and heat. It is well documented that oxygen-limiting conditions in E. coli result in the production of reduced carbon metabolites such as acetate, lactate, and formate (1, 20), which accumulate in high-celldensity cultures and ultimately inhibit further microbial growth. Titers of recombinant proteins in E. coli are limited by several factors including the final cell density (22), the tendency for inclusion...

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