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Updated: May 8, 2026
08:53
A Reporter Based Cellular Assay for Monitoring Splicing Efficiency
Published on: September 15, 2021
Functional premature polyadenylation signals and aberrant splicing within a recombinant protein coding sequence limit
Sujeewa D Wijesuriya1, Robyn L Cotter, Arnold H Horwitz
1XOMA Corporation, 2910 Seventh Street, Berkeley, CA 94710, United States.
Protein Expression and Purification
|September 3, 2013
Summary
Aberrant mRNA processing, including premature polyadenylation and alternative splicing, significantly limited recombinant protein production in CHO-K1 cells. Eliminating these RNA processing signals from the gene enhanced protein expression.
Area of Science:
- Molecular Biology
- Biotechnology
- Protein Expression
Background:
- Recombinant glycoproteins are typically produced at high levels in mammalian cells using strong viral promoters.
- The recombinant complement activator blocking protein, CAB-2 (a fusion of membrane co-factor protein, MCP, and decay accelerating factor, DAF), exhibited unexpectedly low expression in CHO-K1 cell lines.
Purpose of the Study:
- To investigate the causes of low recombinant CAB-2 protein expression in transfected mammalian cells.
- To identify and characterize aberrant mRNA species affecting protein production.
- To determine the impact of internal RNA processing signals on recombinant protein expression.
Main Methods:
- Northern blot analysis to detect CAB-2 mRNA species.
- Reverse transcription polymerase chain reaction (RT-PCR) to identify aberrant splicing.
- 3' rapid amplification of complementary DNA ends (3' RACE) to detect premature polyadenylation.
- Genetic modification of RNA processing elements within the CAB-2 gene.
Main Results:
- Analysis revealed unexpected CAB-2 mRNA species (790 and 1500 bases) in addition to the expected 2300 base species.
- The 1500 base mRNA resulted from aberrant splicing, and the 790 base mRNA resulted from premature polyadenylation within the coding regions.
- Internal polyadenylation signals in native MCP and DAF also generated prematurely polyadenylated mRNAs in human tissues and cell lines.
- Genetic modification to eliminate these signals significantly increased recombinant CAB-2 expression.
Conclusions:
- Aberrant mRNA processing, including splicing and premature polyadenylation, can significantly limit recombinant protein expression.
- Identifying and removing internal RNA processing signals from coding DNA is crucial for maximizing protein production.
- This study highlights the importance of understanding mRNA processing for optimizing gene expression strategies.
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