A novel system for the production of high levels of functional human therapeutic proteins in stable cells with a Semliki Forest virus noncytopathic vector

Casales, Erkuden; Aranda, Alejandro; Quetglas, José I.; Ruiz-Guillen, Marta; Rodríguez-Madoz, Juan R.; Prieto, Jesús; Smerdou, Cristian

doi:10.1016/j.nbt.2010.02.005

Cited by 19 publications

(27 citation statements)

References 47 publications

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“…CT-1 produced by these clones showed a size that was slightly larger than that of recombinant CT-1 expressed in bacteria, indicating that most likely the protein was glycosylated (Fig. 6b), as previously shown [21]. To determine the kinetics of CT-1 expression, selected clones were induced during 5 days with DOX, supernatants were collected every 24 h and secreted CT-1 was measured by specific ELISA.…”

Section: Generation Of Stable Cell Lines Expressing Human Ct-1supporting

confidence: 57%

“…and GFP expression was evaluated by FACS (a) and Western blot with a specific antibody against GFP (b). ctrl stable cell line expressing GFP from an non-inducible RNA-based vector (ncSFV-pac2A-GFP) [21] selective antibiotics. An average of 21.5 clones of each passage from each cell line were sequenced and compared to the parental sequence (Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…BHK-21 cells (ATCC: CCL-10) were cultured in BHK- 21 Glasgow MEM (Gibco BRL, UK) supplemented with 5 % FBS, 10 % tryptose phosphate broth, 2 mM glutamine, 20 mM HEPES, 100 μg/ml streptomycin and 100 IU/ml penicillin (BHK complete medium). Huh7 and HepG2 (ATCC-HB8065) cells were incubated in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10 % FBS, 2 mM glutamine, 100 μg/ ml streptomycin and 100 IU/ml penicillin.…”

Section: Cell Culturementioning

confidence: 99%

“…pShuttle-SFV-Tomato was generated by insertion of the Tomato gene, previously amplified from pCAG-tdTO-MATO-IRES-neo, kindly provided by Dr. Beatriz Pelacho (CIMA), with primers 30 and 31, into BglII and NdeI sites of pShuttle-SFV. To generate pShuttle-SFV-CT, the CT-1 gene was amplified from ncSFV-CT1 [21] by using primers 32 and 33, and the fragment obtained was inserted into BglII and NdeI sites into pShuttle-SFV. To generate pShuttle-SFV-OSM, the coding sequence of human OSM was first obtained by RT-PCR from a sample of CD40-ligand stimulated dendritic cells using primers 34 and 35.…”

Section: Pshuttle-sfv and Pshuttle-sfv-goimentioning

confidence: 99%

“…In order to generate new cell lines in a quick way from the MCL, we also designed a strategy based on switching GFP by the desired transgene using Cre-mediated recombination. This allowed the generation of new cell lines expressing human therapeutic proteins like CT-1, a protein with potent hepatoprotective effects [20], at levels similar to those provided by cell lines generated with ncSFV RNA [21]. In addition, these cells were able to express high levels of toxic proteins like oncostatin M (OSM), a protein that has shown both antiviral and immunostimulatory properties [22], since cells able to express OSM could be grown to the desired amount before being induced.…”

Section: Introductionmentioning

confidence: 99%

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A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system

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proteins contained posttranslational modifications, showed bioactivity, and were efficiently purified. Remarkably, this system allowed production of toxic proteins, like oncostatin-M, since cells able to express it could be grown to the desired amount before induction. These cell lines were easily adapted to growth in suspension, making this methodology very attractive for therapeutic protein production.

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Section: Generation Of Stable Cell Lines Expressing Human Ct-1supporting

confidence: 57%

Section: Resultsmentioning

confidence: 99%

Section: Cell Culturementioning

confidence: 99%

Section: Pshuttle-sfv and Pshuttle-sfv-goimentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system

Aranda

Bezunartea²,

Casales

et al. 2014

Cell. Mol. Life Sci.

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Utilization of sequence variants as biomarkers to analyze population dynamics in cloned cell lines

Lin

Beal

DeGruttola

et al. 2017

Biotech & Bioengineering

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The inherent nature of cloned CHO cell lines includes the presence of genetic and phenotypic drift that leads to heterogeneous populations. The genetic heterogeneity exhibited by these cells can be exploited to understand the population dynamics of cloned cell lines. Understanding the interplay between heterogeneity, cell culture conditions, and population dynamics will allow for critical assessment of overarching cell line development methods and strategies in terms of population and monoclonality. Sequence variants (SVs) are protein isoforms of the gene-of-interest that contain unintended amino acid substitutions, extensions, or truncations that may contribute to heterogeneity. In this case, SVs are unique sequences in the genome of the integrated transgene that can be used as biomarkers to understand the heterogeneity of a monoclonal cell line and how production process conditions can impact population dynamics. In this study, orthogonal genetic and analytical methods were used to examine the variability of SV levels in four different SV-containing cell lines under varied culture conditions and generational ages. Culture conditions tested had little to no impact on SV levels. However, generational age studies showed two distinct trends: stability of SV levels out to approximately 100 generations in cell lines with higher level SVs (>10%) and a progressive decrease of SV levels as the cells age to approximately 100 generations in cell lines with lower level SVs (<10%). The results suggest that the four SV-containing cell lines fall into two distinct population models; SVs present in the whole population of cells and SVs present in only a sub-population of cells. The data presented here are one of the first studies to not only analyze and compare SV levels in both genetic and protein material but also to utilize SVs as biomarkers to probe distinct populations of cloned cell lines. Biotechnol. Bioeng. 2017;114: 1744-1752. © 2017 Wiley Periodicals, Inc.

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Optimization of Protein Expression in Mammalian Cells

et al. 2018

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Recombinant proteins, such as monoclonal antibodies, are produced in mammalian cell lines to introduce proper protein folding and post‐translational modifications, which are essential for full biological activity. In both the industrial and academic environments, the use of recombinant proteins varies widely and, with it, the method of production. The amount of an antibody needed for a toxicity study is far greater than that needed by a research lab performing cellular assays, and the amount of effort put into the development of the protein will vary accordingly. There is no universal strategy for mammalian expression systems, and scientists often struggle to develop a suitable process from the myriad of choices at each step. Here, we elaborate on the various obstacles encountered when planning high‐yield experiments to produce the recombinant proteins of interest. © 2018 by John Wiley & Sons, Inc.

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A novel system for the production of high levels of functional human therapeutic proteins in stable cells with a Semliki Forest virus noncytopathic vector

Cited by 19 publications

References 47 publications

A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system

A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system

Utilization of sequence variants as biomarkers to analyze population dynamics in cloned cell lines

Optimization of Protein Expression in Mammalian Cells

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