Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates

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 acid (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 (94.22 ± 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MW proteins (< 15 kDa). 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.

Transcription factor engineering in CHO cells for recombinant protein production.

The continuous increase of approved biopharmaceutical products drives the development of more efficient recombinant protein expression systems. Chinese hamster ovary (CHO) cells are the mainstay for this purpose but have some drawbacks, such as low levels of expression. Several strategies have been applied to increase the productivity of CHO cells with different outcomes. Transcription factor (TF) engineering has emerged as an interesting and successful approach, as these proteins can act as master regulators; the expression and function of a TF can be controlled by small molecules, and it is possible to design tailored TFs and promoters with desired features. To date, the majority of studies have focused on the use of TFs with growth, metabolic, cell cycle or endoplasmic reticulum functions, although there is a trend to develop new, synthetic TFs. Moreover, new synthetic biological approaches are showing promising advances for the development of specific TFs, even with tailored ligand sensitivity. In this article, we summarize the strategies to increase recombinant protein expression by modulating and designing TFs and with advancements in synthetic biology. We also illustrate how this class of proteins can be used to develop more robust expression systems.

A pilot study of the modulation of sirtuins on arylamine N-acetyltransferase 1 and 2 enzymatic activity.

Arylamine N-acetyltransferase (NAT; E.C. 2.3.1.5) enzymes are responsible for the biotransformation of several arylamine and hydrazine drugs by acetylation. In this process, the acetyl group transferred to the acceptor substrate produces NAT deacetylation and, in consequence, it is susceptible of degradation. Sirtuins are protein deacetylases, dependent on nicotine adenine dinucleotide, which perform post-translational modifications on cytosolic proteins. To explore possible sirtuin participation in the enzymatic activity of arylamine NATs, the expression levels of NAT1, NAT2, SIRT1 and SIRT6 in peripheral blood mononuclear cells (PBMC) from healthy subjects were examined by flow cytometry and Western blot. The in situ activity of the sirtuins on NAT enzymatic activity was analyzed by HPLC, in the presence or absence of an agonist (resveratrol) and inhibitor (nicotinamide) of sirtuins. We detected a higher percentage of positive cells for NAT2 in comparison with NAT1, and higher numbers of SIRT1+ cells compared to SIRT6 in lymphocytes. In situ NAT2 activity in the presence of NAM inhibitors was higher than in the presence of its substrate, but not in the presence of resveratrol. In contrast, the activity of NAT1 was not affected by sirtuins. These results showed that NAT2 activity might be modified by sirtuins.

Heterologous expression of three antigenic proteins from Angiostrongylus cantonensis: ES-7, Lec-5, and 14-3-3 in mammalian cells.

Angiostrongylus cantonensis is a parasitic nematode and the main causative agent of human cerebral eosinophilic meningoencephalitis (EoM). A definitive diagnosis of EoM usually requires serologic or molecular analysis of the patient's clinical sample. Currently, a 31 kDa antigen is used in immunological tests for this purpose, however as a crude antigen preparation it may present cross-reactivity with other helminthic infections, especially echinococcosis. Heterologous expression studies using prokaryotic systems failed on producing antigenic proteins. The aim of this study was to express and purify three recombinant glycoproteins representing A. cantonensis antigens: ES-7, Lec-5, and 14-3-3, in Chinese hamster ovary (CHO) cells and ES-7 in human embryonic kidney (HEK) cells to develop a source of specific antigens to be used in the diagnosis of angiostrongyliasis. The potential diagnostic value of these three proteins was subsequently characterized in one- and two-dimensional electrophoresis and Western blot to dot blot analyses, with Angiostrongylus-positive sera, normal human sera (NHS), and a pool of Echinococcus-positive sera (included as a specificity control) used for detection. In addition, recognition of these three proteins following treatment with N-glycosidase F was examined. The ES-7 proteins that were expressed in HEK and CHO cells, and the Lec-5 protein that was expressed in CHO cells, were specifically recognized by A. cantonensis-positive sera in the 2D electrophoresis analysis. This recognition was shown to be dependent on the presence of glycidic portions, making mammalian cells a very promising source of heterologous expression antigenic proteins from Angiostrongylus.