期刊
ELECTRONIC JOURNAL OF BIOTECHNOLOGY
卷 48, 期 -, 页码 86-94出版社
UNIV CATOLICA DE VALPARAISO
DOI: 10.1016/j.ejbt.2020.09.006
关键词
Cell homogenates; Chinese hamster ovary cells; Precipitation protocols; Protein precipitation; Protein recovery; Protein solubilization; Proteomics; Recombinant proteins; Separation methods
资金
- Consejo Nacional de Ciencia y Tecnologia CONACyT-Mexico [396822]
- Programa de Apoyo a Proyectos de Investigacion e Innovacion Tecnologica, Universidad Nacional Autonoma de Mexico, PAPIIT-UNAM [IN210419, IT-200719, IN-208415]
- Institutional Program of the Instituto de Investigaciones Biomedicas-UNAM: La produccion de biomoleculas de interes biomedico en bacterias y hongos [G0-2019]
Background: Chinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the great importance of precipitation methods for the processing of samples in proteomics, the acetone, methanol-chloroform (M/C), and trichloroacetic add (TCA)-acetone protocols were compared for CHO cells in terms of protein recovery, band pattern resolution, and presence on SDS-PAGE. Results: Higher recovery and similar band profile with cellular homogenates were obtained using acetone precipitation with ultrasonic bath cycles (104.18 +/- 2.67%) or NaOH addition (103.12 +/- 5.74%), compared to the other two protocols tested. TCA-acetone precipitates were difficult to solubilize. which negatively influenced recovery percentage (77.91 +/- 8.79%) and band presence. M/C with ultrasonic homogenization showed an intermediate recovery between the other two protocols (9422 +/- 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MW proteins (<15 k Da). Conclusions: These results help in the processing of samples of CHO cells for their proteomic study by means of an easily accessible, fast protocol, with an almost complete recovery of cellular proteins and the capture of the original complexity of the cellular composition. Acetone protocol could be incorporated to sample-preparation workflows in a straightforward manner and can probably be applied to other mammalian cell lines as well. (C) 2020 Pontificia Universidad Catolica de Valparaiso. Production and hosting by Elsevier B.V. All rights reserved.
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