16 research outputs found
Systematic Single-Cell Analysis of Pichia pastoris Reveals Secretory Capacity Limits Productivity
Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity
Insights into the prevalence and underlying causes of clonal variation through transcriptomic analysis in Pichia pastoris
Cell Surface-Fluorescence Immunosorbent Assay for Real-Time Detection of Hybridomas with Efficient Antibody Secretion at the Single-Cell Level
Array-Based Analysis of Secreted Glycoproteins for Rapid Selection of a Single Cell Producing a Glycoprotein with Desired Glycosylation
High-Throughput Sorting of the Highest Producing Cell via a Transiently Protein-Anchored System
Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring
Assessment of UCOE on Recombinant EPO Production and Expression Stability in Amplified Chinese Hamster Ovary Cells
The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line
Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of the assembled scaffolds with 21 chromosomes isolated by microfluidics to identify chromosomal locations of genes. Furthermore, we investigate genes involved in glycosylation, which affect therapeutic protein quality, and viral susceptibility genes, which are relevant to cell engineering and regulatory concerns. Homologs of most human glycosylation-associated genes are present in the CHO-K1 genome, although 141 of these homologs are not expressed under exponential growth conditions. Many important viral entry genes are also present in the genome but not expressed, which may explain the unusual viral resistance property of CHO cell lines. We discuss how the availability of this genome sequence may facilitate genome-scale science for the optimization of biopharmaceutical protein production