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Over the past decade, several high-value proteins have been produced using plant-based transient expression systems. However, these studies exposed some limitations that must be overcome to allow plant expression systems to reach their full potential. These limitations are the low level of recombinant protein accumulation achieved in some cases, and

Over the past decade, several high-value proteins have been produced using plant-based transient expression systems. However, these studies exposed some limitations that must be overcome to allow plant expression systems to reach their full potential. These limitations are the low level of recombinant protein accumulation achieved in some cases, and lack of efficient co-expression vectors for the production of multi-protein complexes. This study report that tobacco Extensin (Ext) gene 3' untranslated region (UTR) can be broadly used to enhance recombinant protein expression in plants. Extensin is the hydroxyproline-rich glycoprotein that constitutes the major protein component of cell walls. Using transient expression, it was found that the Ext 3' UTR increases recombinant protein expression up to 13.5- and 6-fold in non-replicating and replicating vector systems, respectively, compared to previously established terminators. Enhanced protein accumulation was correlated with increased mRNA levels associated with reduction in read-through transcription. Regions of Ext 3' UTR essential for maximum gene expression included a poly-purine sequence used as a major poly-adenylation site. Furthermore, modified bean yellow dwarf virus (BeYDV)-based vectors designed to allow co-expression of multiple recombinant genes were constructed and tested for their performance in driving transient expression in plants. Robust co-expression and assembly of heavy and light chains of the anti-Ebola virus monoclonal antibody 6D8, as well as E. coli heat-labile toxin (LT) were achieved with the modified vectors. The simultaneous co-expression of three fluoroproteins using the single replicon, triple cassette is demonstrated by confocal microscopy. In conclusion, this study provides an excellent tool for rapid, cost-effective, large-scale manufacturing of recombinant proteins for use in medicine and industry.
ContributorsRosenthal, Sun Hee (Author) / Mason, Hugh (Thesis advisor) / Mor, Tsafrir (Committee member) / Chang, Yung (Committee member) / Arntzen, Charles (Committee member) / Arizona State University (Publisher)
Created2012
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Description
The highly specialized telomerase ribonucleoprotein enzyme is composed minimally of telomerase reverse transcriptase (TERT) and telomerase RNA (TR) for catalytic activity. Telomerase is an RNA-dependent DNA polymerase that syntheizes DNA repeats at chromosome ends to maintain genome stability. While TERT is highly conserved among various groups of species, the TR

The highly specialized telomerase ribonucleoprotein enzyme is composed minimally of telomerase reverse transcriptase (TERT) and telomerase RNA (TR) for catalytic activity. Telomerase is an RNA-dependent DNA polymerase that syntheizes DNA repeats at chromosome ends to maintain genome stability. While TERT is highly conserved among various groups of species, the TR subunit exhibits remarkable divergence in primary sequence, length, secondary structure and biogenesis, making TR identification extremely challenging even among closely related groups of organisms.

A unique computational approach combined with in vitro telomerase activity reconstitution studies was used to identify 83 novel TRs from 10 animal kingdom phyla spanning 18 diverse classes from the most basal sponges to the late evolving vertebrates. This revealed that three structural domains, pseudoknot, a distal stem-loop moiety and box H/ACA, are conserved within TRs from basal groups to vertebrates, while group-specific elements emerge or disappear during animal TR evolution along different lineages.

Next the corn-smut fungus Ustilago maydis TR was identified using an RNA-immunoprecipitation and next-generation sequencing approach followed by computational identification of TRs from 19 additional class Ustilaginomycetes fungi, leveraging conserved gene synteny among TR genes. Phylogenetic comparative analysis, in vitro telomerase activity and TR mutagenesis studies reveal a secondary structure of TRs from higher fungi, which is also conserved with vertebrates and filamentous fungi, providing a crucial link in TR evolution within the opisthokonta super-kingdom.

Lastly, work by collabarotors from Texas A&M university and others identified the first bona fide TR from the model plant Arabidopsis thaliana. Computational analysis was performed to identify 85 novel AtTR orthologs from three major plant clades: angiosperms, gymnosperms and lycophytes, which facilitated phylogenetic comparative analysis to infer the first plant TR secondary structural model. This model was confirmed using site-specific mutagenesis and telomerase activity assays of in vitro reconstituted enzyme. The structures of plant TRs are conserved across land plants providing an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.
ContributorsLogeswaran, Dhenugen (Author) / Chen, Julian J-L (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Borges, Chad R (Committee member) / Arizona State University (Publisher)
Created2019