Multiple gene expression in plants using MIDAS‐P, a versatile type II restriction‐based modular expression vector

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 the 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. Messenger RNA (mRNA) levels of the genes of interest decreased beyond two TUs. Coexpression of the RNA silencing suppressor P19 dramatically increased the 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 four 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.