Abstract
Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30–60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10–60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10–60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.
Highlights
Subcellular fractionation of mammalian cells has been applied for the study of morphology, composition, structure and interactions between organelles [1,2,3], cellular and molecular biology [4] and, more recently, the cell composition through omics approaches [5,6,7]
Since the classical secretion pathway can often become a bottleneck to increase expression of recombinant proteins in Chinese Hamster Ovary (CHO) cells [31,32,33], we focused on the separation of its components by isopycnic centrifugation
Since CHO cells are the preferred host for Enrichment of microsomal organelles for proteomics expression of recombinant proteins in mammalian cells, differential and isopycnic centrifugation protocols were characterized and developed for this cell line
Summary
Subcellular fractionation of mammalian cells has been applied for the study of morphology, composition, structure and interactions between organelles [1,2,3], cellular and molecular biology [4] and, more recently, the cell composition through omics approaches [5,6,7]. As overproducers of recombinant proteins, about 150 published papers have been reported to date that use fractionation protocols oriented only to the isolation of one or few organelles in an adherent phenotype [9,21,22,23,24,25,26] These articles have used wild type and mutant CHO cells for the study of vesicular transport [9,21], lipid composition of plasma membrane (PM) [22], biogenesis of peroxisomes [25], and the subcellular distribution of nsL-TP protein [26]; and for the isolation of Golgi membranes, PM, endoplasmic reticulum (ER), nuclei, mitochondria and lysosomes [23,24]
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