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Effect of Cysteamine on Cell Growth and IgG4 Production in Recombinant Sp2.0 Cells.

ABSTRACT: The manipulation of redox potential in secretory pathway by thiol reducing agents can be a strategy to improve the production levels of disulfide-bonded proteins including recombinant antibodies. Here we have studied the influence of cysteamine on viability and the production level of IgG4 in Sp2.0 cells. For this purpose, the recombinant Sp2.0 cells producing an anti CD33 IgG4, were subjected to different concentrations of cysteamine. At concentrations of 2, 4 and 5 mM cysteamine, the secreted levels of IgG4 did not change significantly. However, in concentration of 7 mM cysteamine, a significant decrease was observed in IgG4 levels which may indicate the cytotoxicity of this compound in higher concentrations. Our results show that the cysteamine treatment reduces the cell viability in a dose-dependent manner. Also it was observed that 2 mM cysteamine had no late effect on IgG4 production level and only at day 3, this concentration of cysteamine decreased the cell viability significantly. To test whether the addition of cysteamine can affect the expression level of protein disulfide isomerase, RT-PCR analysis was carried out. The results revealed that cysteamine does not affect the PDI transcription and expression level of IgG4 in this type of recombinant cells.

SUBMITTER: Jahandar H

PROVIDER: S-EPMC4277631 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Effect of Cysteamine on Cell Growth and IgG4 Production in Recombinant Sp2.0 Cells.

Jahandar Hoda H Vaziri Behrouz B Nematollahi Leila L Afsharirad Tayebeh T Mirabzadeh Esmat E Torkashvand Fatemeh F Khalaj Vahid V

Iranian journal of pharmaceutical research : IJPR 20150101 1

The manipulation of redox potential in secretory pathway by thiol reducing agents can be a strategy to improve the production levels of disulfide-bonded proteins including recombinant antibodies. Here we have studied the influence of cysteamine on viability and the production level of IgG4 in Sp2.0 cells. For this purpose, the recombinant Sp2.0 cells producing an anti CD33 IgG4, were subjected to different concentrations of cysteamine. At concentrations of 2, 4 and 5 mM cysteamine, the secreted ...[more]

PMID: 25561924

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Project description:Sugar nucleotide-dependent (Leloir) glycosyltransferases from plants are important catalysts for the glycosylation of small molecules and natural products. Limitations on their applicability for biocatalytic synthesis arise because of low protein expression (≤10 mg/L culture) in standard microbial hosts. Here, we showed two representative glycosyltransferases: sucrose synthase from soybean and UGT71A15 from apple. A synthetic biology-based strategy of decoupling the enzyme expression from the Escherichia coli BL21(DE3) cell growth was effective in enhancing their individual (approximately fivefold) or combined (approximately twofold) production as correctly folded, biologically active proteins. The approach entails a synthetic host cell, which is able to shut down the production of host messenger RNA by inhibition of the E. coli RNA polymerase. Overexpression of the enzyme(s) of interest is induced by the orthogonal T7 RNA polymerase. Shutting down of the host RNA polymerase is achieved by l-arabinose-inducible expression of the T7 phage-derived Gp2 protein from a genome-integrated site. The glycosyltransferase genes are encoded on conventional pET-based expression plasmids that allow T7 RNA polymerase-driven inducible expression by isopropyl-β- d-galactoside. Laboratory batch and scaled-up (20 L) fed-batch bioreactor cultivations demonstrated improvements in an overall yield of active enzyme by up to 12-fold as a result of production under growth-decoupled conditions. In batch culture, sucrose synthase and UGT71A15 were obtained, respectively, at 115 and 2.30 U/g cell dry weight, corresponding to ∼5 and ∼1% of total intracellular protein. Fed-batch production gave sucrose synthase in a yield of 2,300 U/L of culture (830 mg protein/L). Analyzing the isolated glycosyltransferase, we showed that the improvement in the enzyme production was due to the enhancement of both yield (5.3-fold) and quality (2.3-fold) of the soluble sucrose synthase. Enzyme preparation from the decoupled production comprised an increased portion (61% compared with 26%) of the active sucrose synthase homotetramer. In summary, therefore, we showed that the expression in growth-arrested E. coli is promising for recombinant production of plant Leloir glycosyltransferases.

Dataset Information

Effect of Cysteamine on Cell Growth and IgG4 Production in Recombinant Sp2.0 Cells.

Publications

Effect of Cysteamine on Cell Growth and IgG4 Production in Recombinant Sp2.0 Cells.

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