title: 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.
abstract: 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 (2AE) sequence was further inserted between the intron-containing IFT25 gene and the GFP cDNA, discrete GFP protein was observed to release from the IFT25-2AE-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-IFT25-2AEexpression 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-IFT25-2AEsystem 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.
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Institute of Health Biotechnology, International Collaborative Research Center for Health Biotechnology, College of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Dong
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Institute of Health Biotechnology, International Collaborative Research Center for Health Biotechnology, College of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, China.
authors.forename: He-He
authors.initials: HH
authors.lastname: Hu
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Institute of Health Biotechnology, International Collaborative Research Center for Health Biotechnology, College of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, China.
authors.forename: Zhen-Fang
authors.initials: ZF
authors.lastname: Li
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Institute of Health Biotechnology, International Collaborative Research Center for Health Biotechnology, College of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, China.
authors.forename: Rong-Qiang
authors.initials: RQ
authors.lastname: Cheng
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Institute of Health Biotechnology, International Collaborative Research Center for Health Biotechnology, College of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, China.
authors.forename: De-Mei
authors.initials: DM
authors.lastname: Meng
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Institute of Health Biotechnology, International Collaborative Research Center for Health Biotechnology, College of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, China.
authors.forename: Junping
authors.initials: J
authors.lastname: Wang
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Institute of Health Biotechnology, International Collaborative Research Center for Health Biotechnology, College of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, China. fanzhen@tust.edu.cn.|Obesita & Algaegen LLC, College Station, TX, 77845, USA. fanzhen@tust.edu.cn.|Institute of Health Biotechnology, Rm 334, Yifu Building, Tianjin University of Science and Technology, No. 29 13rd St Tianjin Economic and Technological Development Area, Tianjin, 300457, China. fanzhen@tust.edu.cn.
authors.forename: Zhen-Chuan
authors.initials: ZC
authors.lastname: Fan
mesh_terms: D019149:Bioreactors; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D003062:Codon; D018076:DNA, Complementary; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D019076:Transgenes; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D003062:Codon; D018076:DNA, Complementary; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C057421:virus protein 2A; D049452:Green Fluorescent Proteins
keywords: Bioreactor; Chlamydomonas reinhardtii; FMDV 2A; Intraflagellar transport IFT25 gene; Nuclear transgene expression