A novel bicistronic expression system composed of the intraflagellar transport protein gene ift25 and FMDV 2A sequence directs robust nuclear gene expression in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii offers a great promise for large-scale production of multiple recombinant proteins of pharmaceutical and industrial interest. However, the nuclear-encoding transgenes usually are expressed at a low level, which severely hampers the use of this alga in molecular farming. In...

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Bibliographic Details
Published in:Applied microbiology and biotechnology 2017-05, Vol.101 (10), p.4227-4245
Main Authors: Dong, Bin, Hu, He-He, Li, Zhen-Fang, Cheng, Rong-Qiang, Meng, De-Mei, Wang, Junping, Fan, Zhen-Chuan
Format: Article
Language:English
Subjects:
Algae
Analysis
Antibiotics
Applied Genetics and Molecular Biotechnology
Biomedical and Life Sciences
Bioreactors
Biotechnology
Chlamydomonas reinhardtii
Chlamydomonas reinhardtii - genetics
Codon
Codons
DNA, Complementary
Fluorescence
Foot-and-mouth disease virus
Gene Expression
Genes
Genetic recombination
Genetic research
Genomes
Green Fluorescent Proteins - genetics
Heat shock proteins
Hypocrea jecorina
Life Sciences
Luminescent Proteins - genetics
Microbial Genetics and Genomics
Microbiology
Pharmaceutical sciences
Promoter Regions, Genetic
Protein expression
Recombinant Fusion Proteins - biosynthesis
Recombinant proteins
Studies
Transgenes
Viral Proteins - genetics
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Summary:Chlamydomonas reinhardtii offers a great promise for large-scale production of multiple recombinant proteins of pharmaceutical and industrial interest. However, the nuclear-encoding transgenes usually are expressed at a low level, which severely hampers the use of this alga in molecular farming. In this study, the promoter of the endogenous intraflagellar transport 25 (IFT25) gene of C. reinhardtii was tested for its ability to drive the expression of green fluorescent protein (GFP), which functions as a readout for target gene expression . IFT25 promoter (IFT25P) alone was not able to drive GFP expression to a detectable level. IFT25P, however, can drive robust IFT25-GFP fusion protein expression when the intron-containing IFT25 gene was inserted between IFT25P and GFP cDNA. When an extended version of foot-and-mouth virus 2A protease (2A E ) sequence was further inserted between the intron-containing IFT25 gene and the GFP cDNA, discrete GFP protein was observed to release from the IFT25-2A E -GFP polyprotein via 2A self-cleaving with a cleavage efficacy of approximately 99%. The monomer GFP was accumulated to a level of as high as 0.68% of total soluble proteins. To test whether the newly developed bicistronic IFT25P-IFT25-2A E expression system can be used to overexpress heterologous proteins of different origins and sizes, we inserted codon-optimized cDNAs encoding a Trichoderma reesei xylanase1 (25 kDa) and a Lachnospiraceae bacterium ND2006 type V CRISPR-Cas protein LbCpf1 (147 kDa) to the vector and found that the production of xylanase1 and LbCpf1 was as high as 0.69 and 0.49% of total soluble protein. Our result showed that IFT25P-IFT25-2A E system is more efficient to drive nuclear gene expression in C. reinhardtii than other conventionally used promoters, thus representing a novel efficient recombinant protein expression tool and has the potential to be scaled for commercial production of nuclear-encoded recombinant proteins of different sizes and origins in C. reinhardtii .
ISSN:0175-7598
1432-0614
DOI:10.1007/s00253-017-8177-9