ABSTRACT
For commercial protein therapeutics, Chinese hamster ovary (CHO) cells have an established history of safety, proven capability to express a wide range of therapeutic proteins and high volumetric productivities. Expanding global markets for therapeutic proteins and increasing concerns for broadened access of these medicines has catalyzed consideration of alternative approaches to this platform. Reaching these objectives likely will require an order of magnitude increase in volumetric productivity and a corresponding reduction in the costs of manufacture. For CHO-based manufacturing, achieving this combination of targeted improvements presents challenges. Based on a holistic analysis, the choice of host cells was identified as the single most influential factor for both increasing productivity and decreasing costs. Here we evaluated eight wild-type eukaryotic micro-organisms with prior histories of recombinant protein expression. The evaluation focused on assessing the potential of each host, and their corresponding phyla, with respect to key attributes relevant for manufacturing, namely (a) growth rates in industry-relevant media, (b) adaptability to modern techniques for genome editing, and (c) initial characterization of product quality. These characterizations showed that multiple organisms may be suitable for production with appropriate engineering and development and highlighted that yeast in general present advantages for rapid genome engineering and development cycles.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Eukaryotic Cells/metabolism , Immunologic Factors/biosynthesis , Recombinant Proteins/biosynthesis , Antibodies, Monoclonal/genetics , Biotechnology/methods , Immunologic Factors/genetics , Metabolic Engineering/methods , Recombinant Proteins/genetics , Technology, Pharmaceutical/methodsABSTRACT
In Xenopus embryos, the cell cycle is driven by an autonomous biochemical oscillator that controls the periodic activation and inactivation of cyclin B1-CDK1. The oscillator circuit includes a system of three interlinked positive and double-negative feedback loops (CDK1 -> Cdc25 -> CDK1; CDK1 -/ Wee1 -/ CDK1; and CDK1 -/ Myt1 -/ CDK1) that collectively function as a bistable trigger. Previous work established that this bistable trigger is essential for CDK1 oscillations in the early embryonic cell cycle. Here, we assess the importance of the trigger in the somatic cell cycle, where checkpoints and additional regulatory mechanisms could render it dispensable. Our approach was to express the phosphorylation site mutant CDK1AF, which short-circuits the feedback loops, in HeLa cells, and to monitor cell cycle progression by live cell fluorescence microscopy. We found that CDK1AF-expressing cells carry out a relatively normal first mitosis, but then undergo rapid cycles of cyclin B1 accumulation and destruction at intervals of 3-6 h. During these cycles, the cells enter and exit M phase-like states without carrying out cytokinesis or karyokinesis. Phenotypically similar rapid cycles were seen in Wee1 knockdown cells. These findings show that the interplay between CDK1, Wee1/Myt1, and Cdc25 is required for the establishment of G1 phase, for the normal approximately 20-h cell cycle period, and for the switch-like oscillations in cyclin B1 abundance characteristic of the somatic cell cycle. We propose that the HeLa cell cycle is built upon an unreliable negative feedback oscillator and that the normal high reliability, slow pace and switch-like character of the cycle is imposed by a bistable CDK1/Wee1/Myt1/Cdc25 system.
Subject(s)
CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/metabolism , G1 Phase , Gene Expression Regulation, Developmental , Animals , Cell Cycle , Cell Cycle Proteins/metabolism , Cyclin B/metabolism , Cyclin B1 , Gene Expression Regulation , HeLa Cells , Humans , Mitosis , Models, Biological , Mutation , Nuclear Proteins/metabolism , Protein-Tyrosine Kinases/metabolism , Time Factors , XenopusABSTRACT
A typical protein kinase must recognize between one and a few hundred bona fide phosphorylation sites in a background of approximately 700,000 potentially phosphorylatable residues. Multiple mechanisms have evolved that contribute to this exquisite specificity, including the structure of the catalytic site, local and distal interactions between the kinase and substrate, the formation of complexes with scaffolding and adaptor proteins that spatially regulate the kinase, systems-level competition between substrates, and error-correction mechanisms. The responsibility for the recognition of substrates by protein kinases appears to be distributed among a large number of independent, imperfect specificity mechanisms.
Subject(s)
Protein Kinases/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Consensus Sequence , Forecasting , Humans , Hydrophobic and Hydrophilic Interactions , Mice , Models, Biological , Models, Molecular , NIH 3T3 Cells , Phosphorylation , Protein Binding , Protein Kinases/chemistry , Protein Kinases/classification , Protein Structure, Secondary , Protein Structure, Tertiary , Static Electricity , Substrate SpecificityABSTRACT
The events of cell reproduction are governed by oscillations in the activities of cyclin-dependent kinases (Cdks). Cdks control the cell cycle by catalysing the transfer of phosphate from ATP to specific protein substrates. Despite their importance in cell-cycle control, few Cdk substrates have been identified. Here, we screened a budding yeast proteomic library for proteins that are directly phosphorylated by Cdk1 in whole-cell extracts. We identified about 200 Cdk1 substrates, several of which are phosphorylated in vivo in a Cdk1-dependent manner. The identities of these substrates reveal that Cdk1 employs a global regulatory strategy involving phosphorylation of other regulatory molecules as well as phosphorylation of the molecular machines that drive cell-cycle events. Detailed analysis of these substrates is likely to yield important insights into cell-cycle regulation.
