Heterologous gene expression by infectious and replicon vectors derived from tick-borne encephalitis virus and direct comparison of this flavivirus system with an alphavirus replicon

The flavivirus tick-borne encephaltis virus (TBEV) was established as a vector system for heterologous gene expression. The variable region of the genomic 3′ non-coding region was replaced by an expression cassette consisting of the reporter gene enhanced green fluorescent protein (EGFP) under the translational control of an internal ribosomal entry site element, both in the context of an infectious virus genome and of a replicon lacking the genes of the surface proteins prM/M and E. The expression level and the stability of expression were measured by fluorescence-activated cell-sorting analysis and compared to an established alphavirus replicon vector derived from Venezuelan equine encephaltis virus (VEEV), expressing EGFP under the control of its natural subgenomic promoter. On the first day, the alphavirus replicon exhibited an approximately 180-fold higher expression level than the flavivirus replicon, but this difference decreased to about 20- and 10-fold on days 2 and 3, respectively. Four to six days post-transfection, foreign gene expression by the VEEV replicon vanished almost completely, due to extensive cell killing. In contrast, in the case of the TBEV replicon, the percentage of positive cells and the amount of EGFP expression exhibited only a moderate decline over a time period of almost 4 weeks. The infectious TBEV vector expressed less EGFP than the TBEV replicon at all times. Significant expression from the infectious vector was maintained for four cell-culture passages. The results indicate that the VEEV vector is superior with respect to achieving high expression levels, but the TBEV system may be advantageous for applications that require a moderate, but more enduring, gene expression.

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Production of foreign proteins in the yeast Pichia pastoris

Canadian Journal of Botany ◽

10.1139/b95-336 ◽

1995 ◽

Vol 73 (S1) ◽

pp. 891-897 ◽

Cited By ~ 46

Author(s):

James M. Cregg ◽

David R. Higgins

Keyword(s):

Gene Expression ◽

Pichia Pastoris ◽

Heterologous Gene Expression ◽

Expression System ◽

Methylotrophic Yeast ◽

Culture Broth ◽

Heterologous Gene ◽

Foreign Gene ◽

Heterologous Proteins ◽

Yeast Pichia Pastoris

The methanol-utilizing yeast Pichia pastoris has been developed as a host system for the production of heterologous proteins of commercial interest. An industrial yeast selected for efficient growth on methanol for biomass generation, P. pastoris is readily grown on defined medium in continuous culture at high volume and density. A unique feature of the expression system is the promoter employed to drive heterologous gene expression, which is derived from the methanol-regulated alcohol oxidase I gene (AOX1) of P. pastoris, one of the most efficient and tightly regulated promoters known. The strength of the AOX1 promoter results in high expression levels in strains harboring only a single integrated copy of a foreign-gene expression cassette. Levels may often be further enhanced through the integration of multiple cassette copies into the P. pastoris genome and strategies to construct and select multicopy cassette strains have been devised. The system is particularly attractive for the secretion of foreign-gene products. Because P. pastoris endogenous protein secretion levels are low, foreign secreted proteins often appear to be virtually the only proteins in the culture broth, a major advantage in processing and purification. Key words: heterologous gene expression, methylotrophic yeast, Pichia pastoris, secretion, glycosylation.

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A Dual-Replicon Shuttle Vector System for Heterologous Gene Expression in a Broad Range of Gram-Positive and Gram-Negative Bacteria

Current Microbiology ◽

10.1007/s00284-018-1535-8 ◽

2018 ◽

Vol 75 (10) ◽

pp. 1391-1400

Author(s):

Mingxi Hua ◽

Jingjing Guo ◽

Min Li ◽

Chen Chen ◽

Yuanyuan Zhang ◽

...

Keyword(s):

Gene Expression ◽

Heterologous Gene Expression ◽

Shuttle Vector ◽

Heterologous Gene ◽

Vector System ◽

Gram Negative Bacteria ◽

Gram Positive ◽

Gram Negative

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A shuttle-vector system allows heterologous gene expression in the thermophilic methanogen Methanothermobacter thermautotrophicus ΔH

10.1101/2021.04.20.440605 ◽

2021 ◽

Author(s):

Christian Fink ◽

Sebastian Beblawy ◽

Andreas M. Enkerlin ◽

Lucas Mühling ◽

Largus T. Angenent ◽

...

Keyword(s):

Gene Expression ◽

Positive Selection ◽

Large Scale ◽

Heterologous Gene Expression ◽

Shuttle Vector ◽

Heterologous Gene ◽

Vector System ◽

Physiology And Biochemistry ◽

Methanothermobacter Thermautotrophicus ◽

Power To Gas

AbstractThermophilic Methanothermobacter spp. are used as model microbes to study the physiology and biochemistry of the conversion of hydrogen and carbon dioxide into methane (i.e., hydrogenotrophic methanogenesis), because of their short doubling times and robust growth with high growth yields. Yet, a genetic system for these model microbes was missing despite intense work for four decades. Here, we report the establishment of tools for genetic modification of M. thermautotrophicus. We developed the modular Methanothermobacter vector system, which provided shuttle-vector plasmids (pMVS) with exchangeable selectable markers and replicons for both Escherichia coli and M. thermautotrophicus. For M. thermautotrophicus, a thermostable neomycin-resistance cassette served as the selectable marker for positive selection with neomycin, and the cryptic plasmid pME2001 from Methanothermobacter marburgensis served as the replicon. The pMVS-plasmid DNA was transferred from E. coli into M. thermautotrophicus via interdomain conjugation. After the successful validation of DNA transfer and positive selection in M. thermautotrophicus, we demonstrated heterologous gene expression of a thermostable β-galactosidase-encoding gene (bgaB) from Geobacillus stearothermophilus under the expression control of four distinct synthetic and native promoters. In quantitative in-vitro enzyme activity assays, we found significantly different β-galactosidase activity with these distinct promoters. With a formate dehydrogenase operon-encoding shuttle vector, we allowed growth of M. thermautotrophicus on formate as the sole growth substrate, while this was not possible for the empty vector control. These genetic tools provide the basis to investigate hypotheses from four decades of research on the physiology and biochemistry of Methanothermobacter spp. on a genetic level.Significance StatementThe world economies are facing permanently increasing energy demands. At the same time, carbon emissions from fossil sources need to be circumvented to minimize harmful effects from climate change. The power-to-gas platform is utilized to store renewable electric power and decarbonize the natural gas grid. The microbe Methanothermobacter thermautotrophicus is already applied as the industrial biocatalyst for the biological methanation step in large-scale power-to-gas processes. To improve the biocatalyst in a targeted fashion, genetic engineering is required. With our shuttle-vector system for heterologous gene expression in M. thermautotrophicus, we set the cornerstone to engineer the microbe for optimized methane production, but also for production of high-value platform chemicals in power-to-x processes.

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