RESUMEN
Hydrolysis of polysorbate in biopharmaceutical products has been ascribed to the enzymatic activity from trace levels of residual host cell proteins. In recent years, significant efforts to identify the causative enzymes typically used elaborate, material-intensive and time-consuming approaches. Therefore, the lack of fast and sensitive assays to monitor their activity remains a major bottleneck for supporting process optimization and troubleshooting activities where time and sample throughput are crucial constraints. To address this bottleneck, we developed a novel Electrochemiluminescence-based Polysorbase Activity (EPA) assay to measure hydrolytic activities in biotherapeutics throughout the drug substance manufacturing process. By combining the favorable features of an in-house designed surrogate substrate with a well-established detection platform, the method yields fast (â¼36 h turnaround time) and highly sensitive readouts compatible with high-throughput testing. The assay capability for detecting substrate conversion in a precise and reliable manner was demonstrated by extensive qualification studies and by employing a number of recombinant hydrolases associated with polysorbate hydrolysis. In addition, high assay sensitivity and wide applicability were confirmed for in-process pool samples of three different antibody products by performing a head-to-head comparison between this method and an established liquid chromatography - mass spectrometry based assay for the quantification of free fatty acids. Overall, our results suggest that this new approach is well-suited to resolve differences in hydrolytic activity through all stages of purification.
Asunto(s)
Productos Biológicos , Polisorbatos , Polisorbatos/química , Hidrólisis , Productos Biológicos/química , Cromatografía Liquida , Espectrometría de MasasRESUMEN
Asymmetric ASH1 mRNA transport during mitosis of budding yeast constitutes one of the best-studied examples of mRNA localization. Recently, 2 studies used in vitro motility assays to prove that motile ASH1 mRNA-transport complexes can be reconstituted entirely from recombinant factors. Both studies, however, differed in their conclusions on whether cargo RNA itself is required for particle assembly and thus activation of directional transport. Here we provide direct evidence that stable complexes do assemble in absence of RNA at physiologic conditions and even at ionic strengths above cellular levels. These results directly confirm the previous notion that the ASH1 transport machinery is not activated by the cargo RNA itself, but rather through protein-protein interactions.
Asunto(s)
Regulación Fúngica de la Expresión Génica , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Mitosis , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Concentración Osmolar , Fosforilación , Multimerización de Proteína , Transporte de ARN , ARN de Hongos/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión , Proteínas Represoras/química , Proteínas Represoras/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Asymmetric, motor-protein dependent transport of mRNAs and subsequent localized translation is an important mechanism of gene regulation. Due to the high complexity of such motile particles, our mechanistic understanding of mRNA localization is limited. Over the last two decades, ASH1 mRNA localization in budding yeast has served as comparably simple and accessible model system. Recent advances have helped to draw an increasingly clear picture on the molecular mechanisms governing ASH1 mRNA localization from its co-transcriptional birth to its delivery at the site of destination. These new insights help to better understand the requirement of initial nuclear mRNPs, the molecular basis of specific mRNA-cargo recognition via cis-acting RNA elements, the different stages of RNP biogenesis and reorganization, as well as activation of the motile activity upon cargo binding. We discuss these aspects in context of published findings from other model organisms.
Asunto(s)
Transporte de ARN/genética , Proteínas Represoras/genética , Ribonucleoproteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Unión al ADN/genética , Retículo Endoplásmico/metabolismo , Complejos Multiproteicos/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The assembly and composition of ribonucleic acid (RNA)-transporting particles for asymmetric messenger RNA (mRNA) localization is not well understood. During mitosis of budding yeast, the Swi5p-dependent HO expression (SHE) complex transports a set of mRNAs into the daughter cell. We recombinantly reconstituted the core SHE complex and assessed its properties. The cytoplasmic precomplex contains only one motor and is unable to support continuous transport. However, a defined interaction with a second, RNA-bound precomplex after its nuclear export dimerizes the motor and activates processive RNA transport. The run length observed in vitro is compatible with long-distance transport in vivo. Surprisingly, SHE complexes that either contain or lack RNA cargo show similar motility properties, demonstrating that the RNA-binding protein and not its cargo activates motility. We further show that SHE complexes have a defined size but multimerize into variable particles upon binding of RNAs with multiple localization elements. Based on these findings, we provide an estimate of number, size, and composition of such multimeric SHE particles in the cell.
Asunto(s)
Transporte de ARN/fisiología , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/fisiología , Dimerización , Cadenas Pesadas de Miosina/metabolismo , Cadenas Pesadas de Miosina/fisiología , Miosina Tipo V/metabolismo , Miosina Tipo V/fisiología , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Factores de TranscripciónRESUMEN
Hsc70 is a conserved ATP-dependent molecular chaperone, which utilizes the energy of ATP hydrolysis to alter the folding state of its client proteins. In contrast to the Hsc70 systems of bacteria, yeast and humans, the Hsc70 system of C. elegans (CeHsc70) has not been studied to date.We find that CeHsc70 is characterized by a high ATP turnover rate and limited by post-hydrolysis nucleotide exchange. This rate-limiting step is defined by the helical lid domain at the C-terminus. A certain truncation in this domain (CeHsc70-Δ545) reduces the turnover rate and renders the hydrolysis step rate-limiting. The helical lid domain also affects cofactor affinities as the lidless mutant CeHsc70-Δ512 binds more strongly to DNJ-13, forming large protein complexes in the presence of ATP. Despite preserving the ability to hydrolyze ATP and interact with its cofactors DNJ-13 and BAG-1, the truncation of the helical lid domain leads to the loss of all protein folding activity, highlighting the requirement of this domain for the functionality of the nematode's Hsc70 protein.