Enhancement of Recombinant Protein Production in Transgenic Nicotiana benthamiana Plant Cell Suspension Cultures with Co-Cultivation of Agrobacterium Containing Silencing Suppressors

Ting-Kuo Huang,Bryce Falk,Abhaya Dandekar,Karen Mcdonald

doi:10.3390/ijms19061561

Ting-Kuo Huang, Bryce Falk + Show 2 more

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https://doi.org/10.3390/ijms19061561

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Abstract

We have previously demonstrated that the inducible plant viral vector (CMViva) in transgenic plant cell cultures can significantly improve the productivity of extracellular functional recombinant human alpha-1-antiryspin (rAAT) compared with either a common plant constitutive promoter (Cauliflower mosaic virus (CaMV) 35S) or a chemically inducible promoter (estrogen receptor-based XVE) system. For a transgenic plant host system, however, viral or transgene-induced post-transcriptional gene silencing (PTGS) has been identified as a host response mechanism that may dramatically reduce the expression of a foreign gene. Previous studies have suggested that viral gene silencing suppressors encoded by a virus can block or interfere with the pathways of transgene-induced PTGS in plant cells. In this study, the capability of nine different viral gene silencing suppressors were evaluated for improving the production of rAAT protein in transgenic plant cell cultures (CMViva, XVE or 35S system) using an Agrobacterium-mediated transient expression co-cultivation process in which transgenic plant cells and recombinant Agrobacterium carrying the viral gene silencing suppressor were grown together in suspension cultures. Through the co-cultivation process, the impacts of gene silencing suppressors on the rAAT production were elucidated, and promising gene silencing suppressors were identified. Furthermore, the combinations of gene silencing suppressors were optimized using design of experiments methodology. The results have shown that in transgenic CMViva cell cultures, the functional rAAT as a percentage of total soluble protein is increased 5.7 fold with the expression of P19, and 17.2 fold with the co-expression of CP, P19 and P24.

Highlights

Post-transcriptional gene silencing (PTGS) is a defense mechanism of plants against invading foreign nucleic acids such as viral infection and transgene expression in plant cells [1,2,3]
Recombinant Agrobacterium carrying the viral gene silencing suppressor P19 and the transgenic Cucumber mosaic virus (CMV) inducible viral amplicon (CMViva) cell culture were chosen as a model system to evaluate these co-culture conditions, and to develop the co-cultivation process
The significant enhancement of recombinant human alpha-1-antiryspin (rAAT) production observed in this study suggests that the lower level of rAAT expression observed in the transgenic CMViva cell cultures is partially due to post-transcriptional gene silencing (PTGS); this is consistent with previous studies [22,25,41,42]

Summary

Introduction

Post-transcriptional gene silencing (PTGS) is a defense mechanism of plants against invading foreign nucleic acids such as viral infection and transgene expression in plant cells [1,2,3] In this PTGS process, double-stranded short-interfering RNA (ds siRNA, 21–25 nt) is cleaved from double-stranded RNA (dsRNA) [4] or single-stranded RNA (ssRNA) viral sequences or transgenes by RNase III-type enzyme DICER [5]. The generation of these dsRNA is thought to originate during viral replication, and/or from internal pairing of long RNA assisted by RNA-dependent RNA polymerase (RdRP) [6]. The sequence-complementary mRNA molecules are degraded by the siRNA-RISC complex machinery, and the transgene expression is silenced post-transcriptionally (i.e., mRNA cannot be translated into protein)

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