Clinical manufacturing of recombinant human interleukin 15. I. Production cell line development and protein expression in E. coli with stop codon optimization

Interleukin 15 (IL-15) has shown remarkable biological properties of promoting NK- and T-cell activation and proliferation, as well as enhancing antitumor immunity of CD8(+) T cells in preclinical models. Here, we report the development of an E. coli cell line to express recombinant human Interleukin-15 (rhIL-15) for clinical manufacturing. Human IL-15 cDNA sequence was inserted into a pET28b plasmid and expressed in several E. coli BL21 strains. Through product quality comparisons among several E. coli strains, including E. coli BL21(DE3), BL21(DE3)pLysS, BLR(DE3)pLysS, and BL21-AI, E. coli BL21-AI was selected for clinical manufacturing. Expression optimization was carried out at shake flask and 20-L fermenter scales, and the product was expressed as inclusion bodies that were solubilized, refolded, and purified to yield active rhIL-15. Stop codons of the expression construct were further investigated after 15-20% of the purified rhIL-15 showed an extraneous peak corresponding to an extra tryptophan residue based on peptide mapping and mass spectrometry analysis. It was determined that the presence of an extra tryptophan was due to a stop codon wobble effect, which could be eliminated by replacing TGA (opal) stop codon with TAA (ochre). As a novel strategy, a simple method of demonstrating lack of tRNA suppressors in the production host cells was developed to validate the cells in this study. The E. coli BL21-AI cells containing the rhIL-15 coding sequence with a triplet stop codon TAATAATGA were banked for further clinical manufacturing.     

Self-cycling operation increases productivity of recombinant protein in Escherichia coli

Self-cycling fermentation (SCF), a cyclical, semi-continuous process that induces cell synchrony, was incorporated into a recombinant protein production scheme. Escherichia coli CY15050, a lac(-) mutant lysogenized with temperature-sensitive phage λ modified to over-express β-galactosidase, was used as a model system. The production scheme was divided into two de-coupled stages. The host cells were cultured under SCF operation in the first stage before being brought to a second stage where protein production was induced. In the first stage, the host strain demonstrated a stable cycling pattern immediately following the first cycle. This reproducible pattern was maintained over the course of the experiments and a significant degree of cell synchrony was obtained. By growing cells using SCF, productivity increased 50% and production time decreased by 40% compared to a batch culture under similar conditions. In addition, synchronized cultures induced from the end of a SCF cycle displayed shorter lysis times and a more complete culture-wide lysis than unsynchronized cultures. Finally, protein synthesis was influenced by the time at which the lytic phase was induced in the cell life cycle. For example, induction of a synchronized culture immediately prior to cell division resulted in the maximum protein productivity, suggesting protein production can be optimized with respect to the cell life cycle using SCF.     

A large-scale RNAi screen identifies functional classes of genes shaping synaptic development and maintenance

Neuronal circuit development and function require proper synapse formation and maintenance. Genetic screens are one powerful method to identify the mechanisms shaping synaptic development and stability. However, genes with essential roles in non-neural tissues may be missed in traditional loss-of-function screens. In an effort to circumvent this limitation, we used neuron-specific RNAi knock down in Drosophila and assayed the formation, growth, and maintenance of the neuromuscular junction (NMJ). We examined 1970 Drosophila genes, each of which has a conserved ortholog in mammalian genomes. Knock down of 158 genes in post-mitotic neurons led to abnormalities in the neuromuscular system, including misapposition of active zone components opposite postsynaptic glutamate receptors, synaptic terminal overgrowth and undergrowth, abnormal accumulation of synaptic material within the axon, and retraction of synaptic terminals from their postsynaptic targets. Bioinformatics analysis demonstrates that genes with overlapping annotated function are enriched within the hits for each phenotype, suggesting that the shared biological function is important for that aspect of synaptic development. For example, genes for proteasome subunits and mitotic spindle organizers are enriched among the genes whose knock down leads to defects in synaptic apposition and NMJ stability. Such genes play essential roles in all cells, however the use of tissue- and temporally-restricted RNAi indicates that the proteasome and mitotic spindle organizers participate in discrete aspects of synaptic development. In addition to identifying functional classes of genes shaping synaptic development, this screen also identifies candidate genes whose role at the synapse can be validated by traditional loss-of-function analysis. We present one such example, the dynein-interacting protein NudE, and demonstrate that it is required for proper axonal transport and synaptic maintenance. Thus, this screen has identified both functional classes of genes as well as individual candidate genes that are critical for synaptic development and will be a useful resource for subsequent mechanistic analysis of synapse formation and maintenance.     

When less can yield more – Computational preprocessing of MS/MS spectra for peptide identification

The effectiveness of database search algorithms, such as Mascot, Sequest and ProteinPilot is limited by the quality of the input spectra: spurious peaks in MS/MS spectra can jeopardize the correct identification of peptides or reduce their score significantly. Consequently, an efficient preprocessing of MS/MS spectra can increase the sensitivity of peptide identification at reduced file sizes and run time without compromising its specificity. We investigate the performance of 25 MS/MS preprocessing methods on various data sets and make software for improved preprocessing of mgf/dta‐files freely available from http://hci.iwr.uni‐heidelberg.de/mip/proteomics or http://www.childrenshospital.org/research/steenlab .     

Theoretical and experimental analysis links isoform‐ specific ERK signalling to cell fate decisions

Cell fate decisions are regulated by the coordinated activation of signalling pathways such as the extracellular signal‐regulated kinase (ERK) cascade, but contributions of individual kinase isoforms are mostly unknown. By combining quantitative data from erythropoietin‐induced pathway activation in primary erythroid progenitor (colony‐forming unit erythroid stage, CFU‐E) cells with mathematical modelling, we predicted and experimentally confirmed a distributive ERK phosphorylation mechanism in CFU‐E cells. Model analysis showed bow‐tie‐shaped signal processing and inherently transient signalling for cytokine‐induced ERK signalling. Sensitivity analysis predicted that, through a feedback‐mediated process, increasing one ERK isoform reduces activation of the other isoform, which was verified by protein over‐expression. We calculated ERK activation for biochemically not addressable but physiologically relevant ligand concentrations showing that double‐phosphorylated ERK1 attenuates proliferation beyond a certain activation level, whereas activated ERK2 enhances proliferation with saturation kinetics. Thus, we provide a quantitative link between earlier unobservable signalling dynamics and cell fate decisions.     

Alzheimer's disease: synaptic dysfunction and Aβ

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown cause, characterized by the selective and progressive death of both upper and lower motoneurons, leading to a progressive paralysis. Experimental animal models of the disease may provide knowledge of the pathophysiological mechanisms and allow the design and testing of therapeutic strategies, provided that they mimic as close as possible the symptoms and temporal progression of the human disease. The principal hypotheses proposed to explain the mechanisms of motoneuron degeneration have been studied mostly in models in vitro, such as primary cultures of fetal motoneurons, organotypic cultures of spinal cord sections from postnatal rodents and the motoneuron-like hybridoma cell line NSC-34. However, these models are flawed in the sense that they do not allow a direct correlation between motoneuron death and its physical consequences like paralysis. In vivo, the most widely used model is the transgenic mouse that bears a human mutant superoxide dismutase 1, the only known cause of ALS. The major disadvantage of this model is that it represents about 2%–3% of human ALS. In addition, there is a growing concern on the accuracy of these transgenic models and the extrapolations of the findings made in these animals to the clinics. Models of spontaneous motoneuron disease, like the wobbler and pmn mice, have been used aiming to understand the basic cellular mechanisms of motoneuron diseases, but these abnormalities are probably different from those occurring in ALS. Therefore, the design and testing of in vivo models of sporadic ALS, which accounts for >90% of the disease, is necessary. The main models of this type are based on the excitotoxic death of spinal motoneurons and might be useful even when there is no definitive demonstration that excitotoxicity is a cause of human ALS. Despite their difficulties, these models offer the best possibility to establish valid correlations between cellular alterations and motor behavior, although improvements are still necessary in order to produce a reliable and integrative model that accurately reproduces the cellular mechanisms of motoneuron degeneration in ALS.     

Hidden levels of phylodiversity in Antarctic green algae: further evidence for the existence of glacial refugia

Recent data revealed that metazoans such as mites and springtails have persisted in Antarctica throughout several glacial–interglacial cycles, which contradicts the existing paradigm that terrestrial life was wiped out by successive glacial events and that the current inhabitants are recent colonizers. We used molecular phylogenetic techniques to study Antarctic microchlorophyte strains isolated from lacustrine habitats from maritime and continental Antarctica. The 14 distinct chlorophycean and trebouxiophycean lineages observed point to a wide phylogenetic diversity of apparently endemic Antarctic lineages at different taxonomic levels. This supports the hypothesis that long-term survival took place in glacial refugia, resulting in a specific Antarctic flora. The majority of the lineages have estimated ages between 17 and 84 Ma and probably diverged from their closest relatives around the time of the opening of Drake Passage (30–45 Ma), while some lineages with longer branch lengths have estimated ages that precede the break-up of Gondwana. The variation in branch length and estimated age points to several independent but rare colonization events.