Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

Type 1 protein phosphatase acts in opposition to IpL1 protein kinase in regulating yeast chromosome segregation.

The IPL1 gene is required for high-fidelity chromosome segregation in the budding yeast Saccharomyces cerevisiae. Conditional ipl1ts mutants missegregate chromosomes severely at 37 degrees C. Here, we report that IPL1 encodes an essential putative protein kinase whose function is required during the later part of each cell cycle. At 26 degrees C, the permissive growth temperature, ipl1 mutant cells are defective in the recovery from a transient G2/M-phase arrest caused by the antimicrotubule drug nocodazole. In an effort to identify additional gene products that participate with the Ipl1 protein kinase in regulating chromosome segregation in yeast, a truncated version of the previously identified DIS2S1/GLC7 gene was isolated as a dosage-dependent suppressor of ipl1ts mutations. DIS2S1/GLC7 is predicted to encode a catalytic subunit (PP1C) of type 1 protein phosphatase. Overexpression of the full-length DIS2S1/GLC7 gene results in chromosome missegregation in wild-type cells and exacerbates the mutant phenotype in ipl1 cells. In addition, the glc7-1 mutation can partially suppress the ipl1-1 mutation. These results suggest that type 1 protein phosphatase acts in opposition to the Ipl1 protein kinase in vivo to ensure the high fidelity of chromosome segregation.     

Characterization of a recombinant antibody produced in the course of a high yield fed-batch process

Many mammalian cell fed-batch processes rely on maintaining the cells in a viable and productive state for extended periods of time in order to reach high final concentrations of secreted protein. In the work described herein, a nonamplified NSO cell line was transfected with a vector expressing a recombinant human anti-HIV gp 120 monoclonal antibody (Mab) and a selectable marker, glutamine synthetase. A fed-batch process was developed which improved product yields tenfold over the yields reached in batch culture. In this case, the clone was cultured for a period of 22 days and produced 0.85 g Mab/L. To gauge the effect of extended culture lifetime on product quality, biochemical characteristics of MAb isolated from different time points in the fed-batch culture were determined. The apparent molecular weight of the MAb was constant throughout the course of the culture. Isoelectric focusing revealed four major charged species, with a fifth more acidic species appearing later in the culture. The antigen binding kinetics were constant for MAb isolated throughout the culture period. Glycosylation analysis, on the other hand, revealed that MAb produced later in the culture contained greater percentages of truncated N-acetylglucosamine and highmannose N-glycans. Possible contributions to this underglycosylated material from either cell lysis or synthesis from noviable cells were found to be negligible. Instead, the viable cells appeared to be secreting more truncated and high mannose MAb glycoforms as the culture progressed. (c) 1994 John Wiley & Sons, Inc.     

Action of Brefeldin A on Amphibian Neurons: Passage of Newly Synthesized Proteins Through the Golgi Complex Is Not Required for Continued Fast Organelle Transport in Axons

Abstract: The relation between the availability of newly synthesized protein and lipid and the axonal transport of optically detectable organelles was examined in peripheral nerve preparations of amphibia (Rana catesbeiana and Xenopus laevis) in which intracellular traffic from the endo-plasmic reticulum to the Golgi complex was inhibited with brefeldin A (BFA). Accumulation of fast-transported radio-labeled protein or phospholipid proximal to a sciatic nerve ligature was monitored in vitro in preparations of dorsal root ganglia and sciatic nerve. Organelle transport was examined by computer-enhanced video microscopy of single myelinated axons. BFA reduced the amount of radiolabeled protein and lipid entering the fast-transport system of the axon without affecting either the synthesis or the transport rate of these molecules. The time course of the effect of BFA on axonal transport is consistent with an action at an early step in the intrasomal pathway, and with its action being related to the observed rapid (<1 h) disassembly of the Golgi complex. At a concentration of BFA that reduced fast-transported protein by >95%, no effect was observed on the flux or velocity of anterograde or retrograde organelle transport in axons for at least 20 h. Bidirectional axonal transport of organelles was similarly unaffected following suppression of protein synthesis by >99%. The findings suggest that the anterograde flux of transport organelles is not critically dependent on a supply of newly synthesized membrane precursors. The possibilities are considered that anterograde organelles normally arise from membrane components supplied from a post-Golgi storage pool, as well as from recycled retrograde organelles.     

Dynamics and Distribution of Cyanophages and Their Effect on Marine Synechococcus spp

Cyanophages infecting marine Synechococcus cells were frequently very abundant and were found in every seawater sample along a transect in the western Gulf of Mexico and during a 28-month period in Aransas Pass, Tex. In Aransas Pass their abundance varied seasonally, with the lowest concentrations coincident with cooler water and lower salinity. Along the transect, viruses infecting Synechococcus strains DC2 and SYN48 ranged in concentration from a few hundred per milliliter at 97 m deep and 83 km offshore to ca. 4 x 10 ml near the surface at stations within 18 km of the coast. The highest concentrations occurred at the surface, where salinity decreased from ca. 35.5 to 34 ppt and Synechococcus concentrations were greatest. Viruses infecting strains SNC1, SNC2, and 838BG were distributed in a similar manner but were much less abundant (<10 to >5 x 10 ml). When Synechococcus concentrations exceeded ca. 10 ml, cyanophage concentrations increased markedly (ca. 10 to > 10 ml), suggesting that a minimum host density was required for efficient viral propagation. Data on the decay rate of viral infectivity d (per day), as a function of solar irradiance I (millimoles of quanta per square meter per second), were used to develop a relationship (d = 0.2610I - 0.00718; r = 0.69) for conservatively estimating the destruction of infectious viruses in the mixed layer of two offshore stations. Assuming that virus production balances losses and that the burst size is 250, ca. 5 to 7% of Synechococcus cells would be infected daily by viruses. Calculations based on contact rates between Synechococcus cells and infectious viruses produce similar results (5 to 14%). Moreover, balancing estimates of viral production with contact rates for the farthest offshore station required that most Synechococcus cells be susceptible to infection, that most contacts result in infection, and that the burst size be about 324 viruses per lytic event. In contrast, in nearshore waters, where ca. 80% of Synechococcus cells would be contacted daily by infectious cyanophages, only ca. 1% of the contacts would have to result in infection to balance the estimated virus removal rates. These results indicate that cyanophages are an abundant and dynamic component of marine planktonic communities and are probably responsible for lysing a small but significant portion of the Synechococcus population on a daily basis.     

The Surface Exclusion System of RP1: Investigation of the Roles of trbJ and trbK in the Surface Exclusion, Transfer, and Slow-Growth Phenotypes

The contiguous trbJ and trbK genes of RP1 were cloned individually to study their effects. Surface exclusion was conferred only by trbK and only when gene dosage was high or when trbJ was also present in cis or in trans. This suggests that in the low-copy-number RP1, surface exclusion is due to a two-gene interaction in which trbK is the dominant partner. Among surface exclusion genes, trbJ is novel in yielding a periplasmic product that is also essential for conjugal transfer. This cellular location and the disturbed membrane function that accompanies TrbJ-processing probably accounts for the retarded growth caused by trbJ⁺ clones in Pseudomonas aeruginosa strain PAO.     

Cloning and Expression of a 5-Hydroxytryptamine7 Receptor Positively Coupled to Adenylyl Cyclase

Abstract: In a number of different cell types, phosphorylation of a 63-kDa protein has been shown to increase rapidly in response to stimuli that lead to an increase in intracellular calcium. Here, a stimulus-sensitive protein at this molecular weight is identified in PC12 cells and rat cortical synaptosomes as phosphoglucomutase. In addition, the added phosphate is shown to be in an oligosaccharide terminating in phosphodiester-linked glucose. In synaptosomes, incorporated radioactivity, following incubation with [¹⁴C]glucose or the [β-³⁵S]phosphorothioate analogue of UDP-glucose, was found to increase within 5 s of stimulation and return to baseline within 25 s. Despite the many pathways utilizing glucose, this was the only detectable protein glycosylation observed in synaptosomes. These results indicate that cytoplasmic glycosylation is reversible and rapidly regulated, and suggest that phosphoglucomutase undergoes an alteration in function and/or topography in response to increases in intracellular calcium.     

Isolation and characterization of restriction endonuclease EclHKI from an Enterobacter cloacae strain

An Enterobacter cloacae strain, producing restriction enzyme EclHKI, was isolated from a decaying potato. The enzyme is an isoschizomer of Eam1105I, which recognizes and cleaves GACNNN!NNGTC. EclHKI was produced at high activity (4×10⁴ U/g wet cells) and was purified from contaminants which interfere with restriction digestion by passing the cell lysate through DEAE-Sephacel and heparin columns. Activity was optimal at 37°C in a medium salt buffer.H.-Y. Chan, Y.-C. Chan and P.-C. Shaw are with the Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong: K.-M. Kam is with the Institute of Pathology, Sai Ying Pun Jockey Club Clinic, Hong Kong.     

Monitoring the persistence of Laccaria bicolor as an ectomycorrhizal symbiont of nursery-grown Douglas fir by PCR of the rDNA intergenic spacer

The large-scale inoculation of selected beneficial ectomycorrhizal fungi in forest nurseries has generated renewed interest in the ecology of these symbiotic fungi. However, information on the dissemination and persistence of introduced symbionts is scarce due to the limitation of the current identification methods. To identify ectomycorrhizal fungi on single root tips, we investigated the polymorphism of the PCR-amplified ribosomal DNA intergenic spacer (IGS) from a wide range of ectomycorrhizal fungi. To investigate the reliability of this molecular approach in large-scale surveys, the dissemination and persistence on Douglas fir seedlings of the introduced Laccaria bicolor S238N were assessed in a forest nursery in the Massif Central (France). Several hundred ectomycorrhizas and fruiting bodies were sampled from plots where control and L. bicolor inoculated-Douglas fir seedlings were grown for 1.5 years. PCR typing of mycorrhizas indicated that trees inoculated with L. bicolor S238N remained exclusively colonized by that isolate (or sexually derived isolates) for the entire test period. In contrast, control seedlings were infected by indigenous isolates of Laccaria laccata and Thelephora terrestris. The molecular evidence for the persistence of the introduced mycobiont despite the competition from indigenous isolates of the same species provides further illustration of the potential of exotic species for large-scale microbial application.     

Effect of fructose-1,6-bisphosphate on glutamate uptake and glutamine synthetase activity in hypotix astrocyte cultures

Astrocytes are important in regulating the microencironment of neurons both by catabolic and synthetic pathways. The glutamine synthetase (GS) activity observed in astrocytes affects neurons by removing toxic substances, NH₃ and glutamate; and by providing an important neuronal substrate, glutamine. This glutamate cycle might play a critical role during periods of hypoxia and ischemia, when an increase in extracellular excitatory amino acids is observed. It was previously shown in our laboratory that fructose-1,6-bisphosphate (FBP) protected cortical astrocyte cultures from hypoxic insult and reduced ATP loss following a prolonged (18–30 hrs) hypoxia. In the present study we established the effects of FBP on the level of glutamate uptake and GS activity under normoxic and hypoxic conditions. Under normoxic conditions, [U-¹⁴C]glutamate uptake and glutamine production were independent of FBP treatment; whereas under hypoxic conditions, the initial increase in glutamate uptake and an overall increase in glutamine production in astrocytes were FBP-dependent. Glutamine synthetase activity was dependent on FBP added during the 22 hours of either normoxic- or hypoxic-treatment, hence significant increases in activity were observed due to FBP regardless of the oxygen/ATP levels in situ. These studies suggest that activation of GS by FBP may provide astrocytic protection against hypoxic injury.