Abstract

MIDAS-P is a plant expression vector with blue/white screening for iterative cloning of multiple, tandemly-arranged transcription units (TUs). We have used the MIDAS-P system to investigate expression of up to five genes encoding three anti-HIV proteins and the reporter gene DsRed in Nicotiana benthamiana plants. The anti-HIV cocktail was made up of a broadly neutralizing monoclonal antibody (VRC01), a lectin (Griffithsin), and a single-chain camelid nanobody (J3-VHH). Constructs containing different combinations of 3, 4 or 5 TUs encoding different components of the anti-HIV cocktail were assembled. mRNA levels of the genes of interest decreased beyond two TUs. Co-expression of the RNA silencing suppressor P19 dramatically increased overall mRNA and protein expression levels of each component. The position of individual TUs in 3 TU constructs did not affect mRNA or protein expression levels. However, their expression dropped to non-detectable levels in constructs with 4 or more TUs each containing the same promoter and terminator elements, with the exception of DsRed at the first or last position in 5 TU constructs. This drop was alleviated by co-expression of P19. In short, the MIDAS-P system is suitable for the simultaneous expression of multiple proteins in one construct. This article is protected by copyright. All rights reserved.