Recombinant proteins (r-proteins) are increasingly important in fundamental research and for clinical applications. As many of these r-proteins are of human or animal origin, cultivated mammalian cells are the host of choice to ensure their functional folding and proper posttranslational modifications. Large-scale transfection of human embryonic kidney 293 or Chinese hamster ovary cells is now an established technology that can be used in the production of hundreds of milligram to gram quantities of a r-protein in less than 1 mo from cloning of its cDNA. This chapter aims to provide an overview of large-scale transfection technology with a particular emphasis on calcium phosphate and polyethylenimine-mediated gene transfer.
The potential of Lactobacillus rhamnosus R for producing exopolysaccharide (EPS) when grown on basal minimum medium supplemented with glucose or lactose was investigated. EPS production by L. rhamnosus R is partially growth associated and about 500 mg of EPS per liter was synthesized with both sugars. ؊1 ). It was clearly shown that the amount of EPS produced declined upon prolonged fermentation. Degradation of EPS in fermentation processes was also assessed by measuring its molecular weights and viscosities. As these reductions might have a negative effect on the yield and viscosifying properties of EPS, it was essential to examine possible causes related to this breakdown. The decrease in viscosities and molecular weights of EPS withdrawn at different cultivation times permitted us to suspect the presence of a depolymerizing enzyme in the fermentation medium. Our study on enzymatic production profiles showed a large spectrum of glycohydrolases (␣-D-glucosidase, -D-glucosidase, ␣-D-galactosidase, -D-galactosidase, -D-glucuronidase, and some traces of ␣-L-rhamnosidase). These enzymes were localized, two of them (␣-D-glucosidase and -D-glucuronidase) were partially purified and characterized. When incubated with EPS, these enzymes were capable of lowering the viscosity of the polymer as well as liberating some reducing sugars. Upon prolonged incubation (27 h), the loss of viscosity was increased up to 33%.Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) have generated increasing attention among researchers for the last few years. LAB are food-grade organisms, and the EPS that they produce contribute to the specific rheology and texture of fermented milk products. These EPS represent safe additives for novel food formulations and may have applications in nonfood products (8).There exist three important groups of EPSs produced by LAB: (i) ␣-glucans, mainly composed of ␣-1,6-and ␣-1,3-linked glucose residues, namely, dextrans produced by Leuconostoc mesenteroides subsp. mesenteroides and L. mesenteroides subsp. dextranicum and mutans produced by Streptococcus mutans and S. sobrinus; (ii) fructans, mainly composed of -2,6-linked fructose molecules, such as levan produced by S. salivarius; (iii) heteropolysaccharides produced by mesophilic (Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris) and thermophilic (Lactobacillus delbrueckii subsp. bulgaricus, L. helveticus, and S. thermophilus) LAB (5). The EPS produced by Lactobacillus rhamnosus belong to third group (14). The sugar composition of the EPS produced by L. rhamnosus R studied in this work, as determined on the hydrolysate by high-pressure liquid chromatography (HPLC) and by gas chromatography of the alditol acetates, is the following: Rha, 4; Glc, 2; and Gal, 1 (M. R. Van Casteren, personal communication).A considerable variation can be observed in EPS quantifications. The amount of EPS reported varies from 25 to 132 mg/ liter for L. lactis subsp. cremoris (26) Many studies showed a decrease in the total EPS amount when incuba...
Large-scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins (r-proteins). As many r-proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted, a cost-effective serum-free culture medium that allows their efficient expression and purification is required. In an attempt to design such a serum-free medium, the effect of nine protein hydrolysates on cell proliferation, transfection efficiency, and volumetric productivity was evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphate (SEAP) as reporter genes. The suspension growing, serum-free adapted HEK293SF-3F6 cell line was stably transfected with an EBNA1-expression vector to increase protein expression when using EBV oriP bearing plasmids. Compared to our standard serum-free medium, concomitant addition of the gelatin peptone N3 and removal of BSA slightly enhanced transfection efficiency and significantly increased volumetric productivity fourfold. Using the optimized medium formulation, transfection efficiencies between 40-60% were routinely obtained and SEAP production reached 18 mg/L(-1). To date, we have successfully produced and purified over fifteen r-proteins from 1-14-L bioreactors using this serum-free system. As examples, we describe the scale-up of two secreted his-tagged r-proteins Tie-2 and Neuropilin-1 extracellular domains (ED) in bioreactors. Each protein was successfully purified to >95% purity following a single immobilized metal affinity chromatography (IMAC) step. In contrast, purification of Tie-2 and Neuropilin-1 produced in serum-containing medium was much less efficient. Thus, the use of our new serum-free EBNA1 cell line with peptone-enriched serum-free medium significantly improves protein expression compared to peptone-less medium, and significantly increases their purification efficiency compared to serum-containing medium. This eliminates labor-intensive and expensive chromatographic steps, and allows for the simple, reliable, and extremely fast production of milligram amounts of r-proteins within 5 days posttransfection.
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