Dataset Information

The engineered expression of secreted HSPB5-Fc in CHO cells exhibits cytoprotection in vitro.

ABSTRACT:

Background

HSPB5 is an ATP-independent molecular chaperone that is induced by heat shock or other proteotoxic stresses. HSPB5 is cytoprotective against stress both intracellularly and extracellularly. It acts as a potential therapeutic candidate in ischemia-reperfusion and neurodegenerative diseases.

Results

In this paper, we constructed a recombinant plasmid that expresses and extracellularly secrets a HSPB5-Fc fusion protein (sHSPB5-Fc) at 0.42 μg/ml in CHO-K1 cells. This sHSPB5-Fc protein contains a Fc-tag at the C-terminal extension of HSPB5, facilitating protein-affinity purification. Our study shows that sHSPB5-Fc inhibits heat-induced aggregation of citrate synthase in a time and dose dependent manner in vitro. Administration of sHSPB5-Fc protects lens epithelial cells against cisplatin- or UVB-induced cell apoptosis. It also decreases GFP-Htt^ex1-Q74 insolubility, and reduces the size and cytotoxicity of GFP-Htt^ex1-Q74 aggregates in PC-12 cells.

Conclusion

This recombinant sHSPB5-Fc exhibits chaperone activity to protect cells against proteotoxicity.

SUBMITTER: Li J

PROVIDER: S-EPMC8204567 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Publications

The engineered expression of secreted HSPB5-Fc in CHO cells exhibits cytoprotection in vitro.

Li Jing J Yu Jingjing J Xue Wenxian W Huang Huili H Yan Longjun L Sang Fan F An Shuangshuang S Zhang Jing J Wang Mingli M Zhang Jun J Li Hui H Cui Xiukun X He Jiang J Hu Yanzhong Y

BMC biotechnology 20210614 1

<h4>Background</h4>HSPB5 is an ATP-independent molecular chaperone that is induced by heat shock or other proteotoxic stresses. HSPB5 is cytoprotective against stress both intracellularly and extracellularly. It acts as a potential therapeutic candidate in ischemia-reperfusion and neurodegenerative diseases.<h4>Results</h4>In this paper, we constructed a recombinant plasmid that expresses and extracellularly secrets a HSPB5-Fc fusion protein (sHSPB5-Fc) at 0.42 μg/ml in CHO-K1 cells. This sHSPB5 ...[more]

PMID: 34126963

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Project description:The occurrence of autophagy in recombinant Chinese hamster ovary (rCHO) cell culture has attracted attention due to its effects on therapeutic protein production. Given the significance of glycosylation in therapeutic proteins, this study examined the effects of autophagy-inhibiting chemicals on sialylation of Fc-fusion glycoproteins in rCHO cells. Three chemical autophagy inhibitors known to inhibit different stages were separately treated with two rCHO cell lines that produce the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44. All autophagy inhibitors significantly decreased the sialylation of Fc-fusion glycoprotein in both cell lines. The decrease in sialylation of Fc-fusion glycoprotein is unlikely to be attributed to the release of intracellular enzymes, given the high cell viability and low activity of extracellular sialidases. Interestingly, the five intracellular nucleotide sugars remained abundant in cells treated with autophagy inhibitors. In the mRNA expression profiles of 27 N-glycosylation-related genes using the NanoString nCounter system, no significant differences in gene expression were noted. With the positive effect of supplementing nucleotide sugar precursors on sialylation, attempts were made to enhance the levels of intracellular nucleotide sugars by supplying these precursors. The addition of nucleotide sugar precursors to cultures treated with inhibitors successfully enhanced the sialylation of Fc-fusion glycoproteins compared to the control culture. This was particularly evident under mild stress conditions and not under relatively severe stress conditions, which were characterized by a high decrease in sialylation. These results suggest that inhibiting autophagy in rCHO cell culture decreases sialylation of Fc-fusion glycoprotein by constraining the availability of intracellular nucleotide sugars. KEY POINTS: • The autophagy inhibition in rCHO cell culture leads to a significant reduction in the sialylation of Fc-fusion glycoprotein. • The pool of five intracellular nucleotide sugars remained highly abundant in cells treated with autophagy inhibitors. • Supplementation of nucleotide sugar precursors effectively restores decreased sialylation, particularly under mild stress conditions but not in relatively severe stress conditions.

Project description:Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO) clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean), approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations). Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50 days. Noise minimization may therefore be a novel strategy to reduce apparent expression instability and simplify cell line selection.

Dataset Information

The engineered expression of secreted HSPB5-Fc in CHO cells exhibits cytoprotection in vitro.

Background

Results

Conclusion

Publications

The engineered expression of secreted HSPB5-Fc in CHO cells exhibits cytoprotection in vitro.

Similar Datasets

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