RESUMO
Antibody combination therapeutics (ACTs) are polyvalent biopharmaceuticals that are uniquely suited for the control of complex diseases, including antibiotic resistant infectious diseases, autoimmune disorders and cancers. However, ACTs also represent a distinct manufacturing challenge because the independent manufacture and subsequent mixing of monoclonal antibodies quickly becomes cost prohibitive as more complex mixtures are envisioned. We have developed a virus-free recombinant protein expression platform based on adeno-associated viral (AAV) elements that is capable of rapid and consistent production of complex antibody mixtures in a single batch format. Using both multiplexed immunoassays and cation exchange (CIEX) chromatography, cell culture supernatants generated using our system were assessed for stability of expression and ratios of the component antibodies over time. Cultures expressing combinations of three to ten antibodies maintained consistent expression levels and stable ratios of component antibodies for at least 60 days. Cultures showed remarkable reproducibility following cell banking, and AAV-based cultures showed higher stability and productivity than non-AAV based cultures. Therefore, this non-viral AAV-based expression platform represents a predictable, reproducible, quick and cost effective method to manufacture or quickly produce for preclinical testing recombinant antibody combination therapies and other recombinant protein mixtures.
Assuntos
Dependovirus , Expressão Gênica , Anticorpos de Cadeia Única/biossíntese , Linhagem Celular , Cromatografia por Troca Iônica/métodos , Técnicas de Cocultura , Quimioterapia Combinada , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/isolamento & purificaçãoRESUMO
In this issue of Molecular Cell, Jani et al. (2009) present the crystal structure of a region of TREX2, thereby providing a physical framework for how the transcription and export machineries might cooperate to "gate" genes to the nuclear pore.
Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ciclo Celular/química , Poro Nuclear/metabolismo , Proteínas Nucleares/química , Transporte de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/fisiologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , Poro Nuclear/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Porinas , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
We used a quantitative, high-density genetic interaction map, or E-MAP (Epistatic MiniArray Profile), to interrogate the relationships within and between RNA-processing pathways. Due to their complexity and the essential roles of many of the components, these pathways have been difficult to functionally dissect. Here, we report the results for 107,155 individual interactions involving 552 mutations, 166 of which are hypomorphic alleles of essential genes. Our data enabled the discovery of links between components of the mRNA export and splicing machineries and Sem1/Dss1, a component of the 19S proteasome. In particular, we demonstrate that Sem1 has a proteasome-independent role in mRNA export as a functional component of the Sac3-Thp1 complex. Sem1 also interacts with Csn12, a component of the COP9 signalosome. Finally, we show that Csn12 plays a role in pre-mRNA splicing, which is independent of other signalosome components. Thus, Sem1 is involved in three separate and functionally distinct complexes.
Assuntos
Mapeamento de Interação de Proteínas , Processamento Pós-Transcricional do RNA , Splicing de RNA , Transporte de RNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complexo do Signalossomo COP9 , Exorribonucleases , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
We recently reported that the 'hydrophobic patch' (HP) of the Saccharomyces cerevisiae S-phase cyclin Clb5 facilitates its interaction with Orc6 (via its Cy or RXL motif), providing a mechanism that helps prevent rereplication from individual origins. This is the first finding of a biological function for an interaction between a cyclin and a cyclin-binding motif (Cy or RXL motif) in a target protein in Saccharomyces cerevisiae. It is also the first such example involving a B-type cyclin in any organism. Yet, some of our observations as well as work from other groups suggest that HP-RXL interactions are functionally important for cyclin-Cdk signaling to other targets. The evolutionary conservation of the HP motif suggests that it allows cyclins to carry out important and specialized functions.
Assuntos
Ciclina B/fisiologia , Regulação Fúngica da Expressão Gênica , Complexo de Reconhecimento de Origem/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Ciclo Celular/genética , Ciclo Celular/fisiologia , Ciclina B/química , Ciclina B/genética , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/fisiologia , Ciclinas/genética , Ciclinas/fisiologia , Replicação do DNA , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/genética , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Cyclin-dependent kinases are critical regulators of eukaryotic DNA replication. We show that the S-phase cyclin Clb5 binds stably and directly to the origin recognition complex (ORC). This interaction is mediated by an "RXL" target sequence, or "Cy" motif, in the Orc6 subunit that is recognized by the "hydrophobic patch" region on Clb5. The Clb5-Orc6 interaction requires replication initiation, and is maintained throughout the remainder of S phase and into M phase. Eliminating the Clb5-Orc6 interaction has no effect on initiation of replication but instead sensitizes cells to lethal overreplication. We propose that Clb5 binding to ORC provides an origin-localized replication control switch that specifically prevents reinitiation at replicated origins.
Assuntos
Ciclina B/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ciclina B/química , Ciclina B/genética , Replicação do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Estabilidade Enzimática , Mutação , Complexo de Reconhecimento de Origem , Fase S , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
The minimal requirements for a eukaryotic origin of replication are an initiator binding site and a region of helically unstable DNA [DNA unwinding element (DUE)]. Budding yeast origins consist of modular elements, and one of these elements, B2, has been proposed to act as a DUE. To test this hypothesis, we screened for sequences that function at the B2 element of ARS1. We found that the B2 element required A-rich sequences, but that the function of these identified sequences did not correlate with helical instability. Instead, the sequences that substituted fully for B2 function showed similarity to the ARS consensus sequence (ACS). The ACS is the binding site for the initiator origin recognition complex (ORC), but the selected sequences are not strong ORC binding sites in vitro. Nonfunctional B2 sequences show a corresponding loss in Mcm2-7p origin association. The function of these mutant sequences is rescued by Cdc6p overexpression. We propose that the B2 element requires specific sequences to bind a component of the pre-RC.
