Dataset Information

Design, construction, and expression of recombinant human interferon beta gene in CHO-s cell line using EBV-based expression system.

ABSTRACT: Background and purpose:Codon optimization has been considered as a powerful strategy to increase the expression level of protein therapeutics in mammalian cells. As an empirical approach to study the effects of the codon usage and GC content on heterologous gene expression in suspension adapted Chinese hamster ovary (CHO-s) cells, we redesigned the recombinant human interferon beta (rhIFN- ?) gene based on the codon preference of the CHO cell in a way to increase the GC content in the third position of each codon. Experimental approach:The nucleotide sequence of the codon-optimized rhIFN-? was synthesized in parallel with the wild-type and expressed transiently in CHO-s cells using Epstein-Bar virus (EBV)-based expression system. The protein expression of the rhIFN-? by codon-optimized and wild-type genes were quantified using ELISA test. Findings / Results:The results indicated a 2.8-fold increase in the expression level of the biologically active form of the rhIFN-? by codon-optimized sequence. Conclusion and implications:These results shed light on the capability of codon optimization to create a stable CHO cell for scaling up the production of recombinant therapeutics such as rhIFN-?.

SUBMITTER: Shayesteh M

PROVIDER: S-EPMC7306247 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Design, construction, and expression of recombinant human interferon beta gene in CHO-s cell line using EBV-based expression system.

Shayesteh Mohadeseh M Ghasemi Fahimeh F Tabandeh Fatemeh F Yakhchali Bagher B Shakibaie Mehdi M

Research in pharmaceutical sciences 20200401 2

<h4>Background and purpose</h4>Codon optimization has been considered as a powerful strategy to increase the expression level of protein therapeutics in mammalian cells. As an empirical approach to study the effects of the codon usage and GC content on heterologous gene expression in suspension adapted Chinese hamster ovary (CHO-s) cells, we redesigned the recombinant human interferon beta (rhIFN- β) gene based on the codon preference of the CHO cell in a way to increase the GC content in the th ...[more]

PMID: 32582354

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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.

Project description:Chinese hamster ovary (CHO) cell lines represent the most commonly used mammalian expression system for the production of therapeutic proteins. In this context, detailed knowledge of the CHO cell transcriptome might help to improve biotechnological processes conducted by specific cell lines. Nevertheless, very few assembled cDNA sequences of CHO cells were publicly released until recently, which puts a severe limitation on biotechnological research. Two extended annotation systems and web-based tools, one for browsing eukaryotic genomes (GenDBE) and one for viewing eukaryotic transcriptomes (SAMS), were established as the first step towards a publicly usable CHO cell genome/transcriptome analysis platform. This is complemented by the development of a new strategy to assemble the ca. 100 million reads, sequenced from a broad range of diverse transcripts, to a high quality CHO cell transcript set. The cDNA libraries were constructed from different CHO cell lines grown under various culture conditions and sequenced using Roche/454 and Illumina sequencing technologies in addition to sequencing reads from a previous study. Two pipelines to extend and improve the CHO cell line transcripts were established. First, de novo assemblies were carried out with the Trinity and Oases assemblers, using varying k-mer sizes. The resulting contigs were screened for potential CDS using ESTScan. Redundant contigs were filtered out using cd-hit-est. The remaining CDS contigs were re-assembled with CAP3. Second, a reference-based assembly with the TopHat/Cufflinks pipeline was performed, using the recently published draft genome sequence of CHO-K1 as reference. Additionally, the de novo contigs were mapped to the reference genome using GMAP and merged with the Cufflinks assembly using the cuffmerge software. With this approach 28,874 transcripts located on 16,492 gene loci could be assembled. Combining the results of both approaches, 65,561 transcripts were identified for CHO cell lines, which could be clustered by sequence identity into 17,598 gene clusters.

Dataset Information

Design, construction, and expression of recombinant human interferon beta gene in CHO-s cell line using EBV-based expression system.

Publications

Design, construction, and expression of recombinant human interferon beta gene in CHO-s cell line using EBV-based expression system.

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