Development of Tomato bushy stunt virus-based vectors for fusion and non-fusion expression of heterologous proteins in an alternative host Nicotiana excelsiana.

Plant virus-based expression systems are an alternative expression platform for the production of clinically and industrially useful recombinant proteins. Nonetheless, due to a lack of viral vector with the commercial potentials, it is urgent to design and develop new, versatile, and efficient plant virus vectors. The genome of Tomato bushy stunt virus (TBSV) offers an attractive alternative to being modified as a vector for producing heterologous proteins in plants. Here, we developed a set of novel fusion and non-fusion TBSV-CP replacement vectors, which provide more flexible and efficient tools for expressing proteins of interest in plants. An alternative tobacco plant, Nicotiana excelsiana, was used in this study as a host for newly constructed TBSV vectors because the unwanted necrotic effects were reported on the commonly used Nicotiana benthamiana host associated with expression of TBSV-encoded P19 protein. The data showed that TBSV vectors caused a symptomless infection and overexpressed reporter gene in N. excelsiana leaves, demonstrating that N. excelsiana is an ideal host plant for TBSV-mediated heterologous gene expression. Moreover, a TBSV non-fusion vector, dAUG, shows the similar accumulation level of reporter proteins to that of TMV- and PVX-based vectors in side-by-side comparison and provides more flexible aspects than the previously developed TBSV vectors. Collectively, our newly developed TBSV expression system adds a new member to the family of plant viral expression vectors and meanwhile offers a flexible and highly effective approach for producing proteins of interest in plants. KEY POINTS: • The TBSV-based transient expression system has been significantly improved. • The necrotic effects caused by viral P19 protein were avoided by the usage of N. excelsiana as a host plant. • The expression level of the non-fusion vector was similar to the most effective virus vectors reported so far.

Purification of the recombinant green fluorescent protein from tobacco plants using alcohol/salt aqueous two-phase system and hydrophobic interaction chromatography.

BACKGROUND: The green fluorescent protein (GFP) has been regarded as a valuable tool and widely applied as a biomarker in medical applications and diagnostics. A cost-efficient upstream expression system and an inexpensive downstream purification process will meet the demands of the GFP protein with high-purity. RESULTS: The recombinant GFP was transiently expressed in an active form in agoinoculated Nicotiana benthamiana leaves by using Tobacco mosaic virus (TMV) RNA-based overexpression vector (TRBO). The yield of recombinant GFP was up to ~ 60% of total soluble proteins (TSP). Purification of recombinant GFP from the clarified lysate of N. benthaniana leaves was achieved by using an alcohol/salt aqueous two-phase system (ATPS) and following with a further hydrophobic interaction chromatography (HIC). The purification process takes only ~ 4 h and can recover 34.1% of the protein. The purity of purified GFP was more than 95% and there were no changes in its spectroscopic characteristics. CONCLUSIONS: The strategy described here combines the advantages of both the economy and efficiency of plant virus-based expression platform and the simplicity and rapidity of environmentally friendly alcohol/salt ATPS. It has a considerable potential for the development of a cost-efficient alternative for production of recombinant GFP.

Molecular gated-AlGaN/GaN high electron mobility transistor for pH detection.

A molecular gated-AlGaN/GaN high electron mobility transistor has been developed for pH detection. The sensing surface of the sensor was modified with 3-aminopropyltriethoxysilane to provide amphoteric amine groups, which would play the role of receptors for pH detection. On modification with 3-aminopropyltriethoxysilane, the transistor exhibits good chemical stability in hydrochloric acid solution and is sensitive for pH detection. Thus, our molecular gated-AlGaN/GaN high electron mobility transistor acheived good electrical performances such as chemical stability (remained stable in hydrochloric acid solution), good sensitivity (37.17 µA/pH) and low hysteresis. The results indicate a promising future for high-quality sensors for pH detection.

Co-expression With Replicating Vector Overcoming Competitive Effects Derived by a Companion Protease Inhibitor in Plants.

Plant-based expression platforms are currently gaining acceptance as a viable alternative for the production of recombinant proteins (RPs), but the degradation of RPs by proteases in cells hinders their superb potentials. Co-expression of a protease inhibitor (PI) shows promise as a strategy to prevent RP from proteolytic degradation in plants. However, competitive effects behind the PI-RP co-expression system may mask or obfuscate the in situ protective effects of a companion PI. Here, we explored the competitive effects by co-expressing reteplase (rPA) with three unrelated PIs, namely NbPR4, HsTIMP, and SlCYS8, in Nicotiana benthamiana leaves. Remarkably, the accumulation of rPA was significantly repressed by each of the three PIs, suggesting that the competitive effects may be common among the PIs. The repression can be attenuated by reducing the PI inoculum dose in the co-inoculation mixtures, showing a negative correlation between the PI abundance of the PI-RP system and competitive effects. Interestingly, when a replicating vector was used to modulate the relative abundance of PI and RP in vivo, rPA was still boosted even at the maximal testing dose of PI, indicating that the competitive effects reduced to an ignorable level by this in vivo approach. Furthermore, a 7- to 12-fold increase of rPA was achieved, proving that it is a useful way for stimulating the potentials of a companion PI by overcoming competitive effects. And, this approach can be applied to molecular farming for improving the RP yields of plant expression systems.

Molecular diversity and differential expression of starch-synthesis genes in developing kernels of three maize inbreds.

The maize genome remains abundant in molecular diversity, and the rich genetic diversity of maize starch-synthesis genes is crucial for controlling various grain traits. To explore the unique mechanism controlling the advantageous waxy trait and characterize the molecular feature of genes relevant to starch composition in two elite waxy inbreds, expression profiling combined with gene organization analysis was performed in them as compared to one normal inbred. Genotype-specific expression patterns were observed for most genes studied. The waxy inbreds were shown to contain mutations in multiple starch-synthesis genes, namely gbssI (wx), gbssIIb and isa2 (potentially isa3 too).The mis-splicing events directly accounted for wx loss of function. Contrarily, disruption of 5' and 3' transcript sequence may contribute to the absence of GbssIIb and Isa2 transcripts in waxy inbreds, respectively. Besides, the splicing of Sugary1 transcript was developmentally regulated in the normal inbred, and DNA polymorphisms were detected within SSIIIb-1 gene in waxy inbreds.

[Transient expression of bioactive recombinant human plasminogen activator in tobacco leaf].

OBJECTIVE: To assess the potential of transient expression of recombinant human plasminogen activator (rhPA) in plants as a cost-effective approach for recombinant rhPA production. METHODS: Tobacco mosaic virus-based expression vector pTMV rhPA-NSK and plant binary expression vector pJ Zera-rhPA were constructed by in vitro sequence synthesis and subcloning. The two vectors were inoculated on either Nicotiana benthamiana or N. excelsiana leaves via agroinfiltration. The expression of recombinant rhPA in Nicotiana leaves was examined using Western blotting and ELISA, and the in vitro fibrinolysis activity of plant-produced rhPA was assessed by fibrin agarose plate assay (FAPA). RESULTS: Five to nine days after infiltration with an Agrobacterium inoculum containing pTMV rhPA-NSK, necrosis appeared in the infiltrated area on the leaves of both Nicotiana plants, but intact recombinant rhPA was still present in the necrotic leaf tissues. The accumulation level of recombinant rhPA in infiltrated N. benthamiana leaves was significantly higher than that in N. excelsiana leaves (P < 0.05). The yield of recombinant rhPA was up to 0.6% of the total soluble protein (or about 60.0 µg per gram) in the fresh leaf biomass at 7 days post-inoculation. The plant-derived rhPA was bioactive to convert inactive plasminogen to active plasmin. No necrosis occurred in pJ Zera-rhPA-infiltrated leaves. The Zera-rhPA protein was partially cleaved between the site of Zera tag and rhPA sequence in both Nicotiana leaves. We speculated that the formation of Zera tags-induced particles in the plant cells was a dynamic process of progressive aggregation in which some of the soluble polypeptides were encapsulated in these particles. CONCLUSIONS: Enzymatically active recombinant rhPA can be rapidly expressed in tobacco plants using the plant viral ampliconbased system, which offers a promising alternative for cost-effective production of recombinant rhPA.

[Effect of the hydrophobin HFBI-fusion tag on exogenous protein accumulation in tobacco plant].

OBJECTIVE: To explore the mechanisms by which HFBI fusions increase recombinant fusion protein accumulation in plants. METHODS: The HFBI sequence from Trichoderma reesei was synthesized and two plant expression vectors for expression of green fluorescence protein (GFP) and GFP-HFBI were constructed. The vectors were inoculated in Nicotiana benthamiana plants through agroinfiltration, and the expression levels and mRNA accumulation levels of GFP in Nicotiana leaves were examined by Western blotting, ELISA and RT-PCR. RESULTS: The HFBI fusion tag significantly enhanced the accumulation of GFP in the leaves of N. benthamiana without causing toxic effects. Endoplasmic reticulum-targeted GFP-HFBI fusion induced the formation of spherical protein particles in the plant cells. CONCLUSION: HFBI fusions can increase the accumulation of its fusion partner in plants by forming stable protein particles, which probably shields the target protein from endogenous protease-induced degadation. HFBI fusion technology provides an alternative to improving recombinant protein expression in plants from agroinfection-compatible expression vectors.

AtCopeg1, the unique gene originated from AtCopia95 retrotransposon family, is sensitive to external hormones and abiotic stresses.

Retrotransposons, the important component of eukaryotic genome, are seeds of evolution and play great role in creating new genes. The compact Arabidopsis genome harbors over 200 Copia-like retrotransposons, but mostly silent. Here we isolated an expressed gene AtCopeg1 (Copia evolved gene 1), which shows higher than 90% identity to AtCopia95_I, the consensus sequence encoding AtCopia95 polyprotein. AtCopeg1 is the unique gene evolved from AtCopia95 family. It is an intron-containing gene with two alternative 3' ends. The transcript accumulation of AtCopeg1 is tissue-specific, also significantly affected by external hormones and abiotic stresses. The presence of regulatory elements in its promoter region (originating from AtCopia95_I and AtCopia95 long terminal repeat), is adequate for conferring its essential expression feature. Thus, AtCopeg1 is a versatile functional gene involved in many developmental and adaptive processes probably including the signaling crosstalk of hormone and nutrient stress. Our work highlighted the role of transposable elements in creating new functional genes, and will incite the enthusiasm for isolation and functional characterization of plant genes evolved from those previously considered as selfish and junk DNA.