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Improvement of the transient expression system for production of recombinant proteins in plants.

ABSTRACT: An efficient and high yielding expression system is required to produce recombinant proteins. Furthermore, the transient expression system can be used to identify the localization of proteins in plant cells. In this study, we demonstrated that combination of a geminiviral replication and a double terminator dramatically enhanced the transient protein expression level in plants. The GFP protein was expressed transiently in lettuce, Nicotiana benthamiana, tomatoes, eggplants, hot peppers, melons, and orchids with agroinfiltration. Compared to a single terminator, a double terminator enhanced the expression level. A heat shock protein terminator combined with an extensin terminator resulted in the highest protein expression. Transiently expressed GFP was confirmed by immunoblot analysis with anti-GFP antibodies. Quantitative analysis revealed that the geminiviral vector with a double terminator resulted in the expression of at least 3.7?mg/g fresh weight of GFP in Nicotiana benthamiana, approximately 2-fold that of the geminiviral vector with a single terminator. These results indicated that combination of the geminiviral replication and a double terminator is a useful tool for transient expression of the gene of interest in plant cells.

SUBMITTER: Yamamoto T

PROVIDER: S-EPMC5859073 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Improvement of the transient expression system for production of recombinant proteins in plants.

Yamamoto Tsuyoshi T Hoshikawa Ken K Ezura Kentaro K Okazawa Risa R Fujita Satoshi S Takaoka Miyo M Mason Hugh S HS Ezura Hiroshi H Miura Kenji K

Scientific reports 20180319 1

An efficient and high yielding expression system is required to produce recombinant proteins. Furthermore, the transient expression system can be used to identify the localization of proteins in plant cells. In this study, we demonstrated that combination of a geminiviral replication and a double terminator dramatically enhanced the transient protein expression level in plants. The GFP protein was expressed transiently in lettuce, Nicotiana benthamiana, tomatoes, eggplants, hot peppers, melons, ...[more]

PMID: 29555968

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Project description:BACKGROUND:Corynebacterium glutamicum is a traditional food-grade industrial microorganism, in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantage of low recombinant protein expression level is still difficult to be solved. Here, according to the bacteria-specific polycistronic feature that multiple proteins can be translated in one mRNA, efforts have been made to insert a leading peptide gene upstream of target genes as an expression enhancer, and it is found that this can remarkably improve the expression level of proteins under the control of inducible tac promoter in C. glutamicum. RESULTS:In this research, the Escherichia coli (E. coli) tac promoter combined with 24 different fore-cistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic expression vectors were isolated and exhibited high efficiency under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins- aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-? (BoIFN-?), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type ? N-terminal peptide (P?NP). All examined proteins were highly expressed compared with the original vector with tac promoter. Large-scale production of P?NP was also performed in fed-batch cultivation, and the highest P?NP production level was 1.2 g/L. CONCLUSION:In this study, the strength of the inducible tac promoter for C. glutamicum was improved by screening and inserting fore-cistron sequences in front of the target genes. Those vectors with bicistronic expression patterns have strong compatibility for expressing various heterogeneous proteins in high yield. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.

Dataset Information

Improvement of the transient expression system for production of recombinant proteins in plants.

Publications

Improvement of the transient expression system for production of recombinant proteins in plants.

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