Development of a transient, high-level expression platform for protein production in plants

Plants are an attractive alternative to conventional expression systems for the production of recombinant proteins and useful biologics, however, the economic viability of plant made proteins is strongly yield dependent. This study aimed to improve transgene expression levels in the plant host Nicotiana benthamiana using the Agroinfiltration transient expression platform. Independent investigation of the physical, chemical and genetic features associated with Agroinfiltration identified factors that improved transformation frequencies, elevated transgene expression levels and ultimately improved protein yield. The major outcome of this research was a novel hyper-expression system for biofarming recombinant proteins in plants

Improving agroinfiltration-based transient gene expression in Nicotiana benthamiana

Abstract Background Agroinfiltration is a simple and effective method of delivering transgenes into plant cells for the rapid production of recombinant proteins and has become the preferred transient expression platform to manufacture biologics in plants. Despite its popularity, few studies have sought to improve the efficiency of agroinfiltration to further increase protein yields. This study aimed to increase agroinfiltration-based transient gene expression in Nicotiana benthamiana by improving all levels of transgenesis. Results Using the benchmark pEAQ-HT deconstructed virus vector system and the GUS reporter enzyme, physical, chemical, and molecular features were independently assessed for their ability to enhance Agrobacterium-mediated transformation and improve protein production capacities. Optimal Agrobacterium strain, cell culture density and co-cultivation time for maximal transient GUS (β-glucuronidase) expression were established. The effects of chemical additives in the liquid infiltration media were investigated and acetosyringone (500 μM), the antioxidant lipoic acid (5 μM), and a surfactant Pluronic F-68 (0.002%) were all shown to significantly increase transgene expression. Gene products known to suppress post-transcriptional gene silencing, activate cell cycle progression and confer stress tolerance were also assessed by co-expression. A simple 37 °C heat shock to plants, 1–2 days post infiltration, was shown to dramatically increase GUS reporter levels. By combining the most effective features, a dual vector delivery system was developed that provided approximately 3.5-fold higher levels of absolute GUS protein compared to the pEAQ-HT platform. Conclusions In this paper, different strategies were assessed and optimised with the aim of increasing plant-made protein capacities in Nicotiana benthamiana using agroinfiltration. Chemical additives, heat shock and the co-expression of genes known to suppress stress and gene silencing or stimulate cell cycle progression were all proven to increase agroinfiltration-based transient gene expression. By combining the most effective of these elements a novel expression platform was developed capable of producing plant-made protein at a significantly higher level than a benchmark hyper-expression system

Characterisation of Sweet potato collusive virus (SPCV) isolates from sweet potato (Ipomea batatas) in Australia

Sweet potato collusive virus (SPCV) is a member of the genus Cavemovirus, family Caulimoviridae, for which only one full-length genome sequence has been reported. SPCV was first detected in Australia in 2007 in two sweet potato accessions using the nitrocellulose membrane ELISA kit developed by the International Potato Centre (CIP). Infected plants were also shown to contain isometric virions of ~ 50 nm, typical of members of the genera Cavemovirus, Caulimovirus, Petuvirus and Soymovirus. We have now sequenced and characterised the complete genomes of the two SPCV isolates (designated SPCV-Aus1 and -Aus2) using a combination of next-generation sequencing and PCR/Sanger sequencing. The sequences of both isolates encode three major ORFs with a genome organisation typical of cavemoviruses. However, isolate SPCV-Aus2 possesses a considerably shorter genome length of 7275 bp compared to SPCV-Aus1 (7712 bp) and the only available full-length sequence from a Portuguese isolate (7723 bp; GenBank accession number NC_015328). Further, ORF 1 of SPCV-Aus2 is considerably shorter than the ORF 1 length of both SPCV-Aus1 and SPCV-Mad1. Phylogenetic and PASC analysis showed that SPCV-Aus1 is closely related to SPCV isolates from North and Central America, whereas SPCV-Aus2 clustered together with isolates from Portugal and Africa