Takara Human Cell-Free Protein Expression Master Mix For Mrna

Human Cell-Free Protein Expression Master Mix for mRNA is a cell-free protein synthesis reagent that contains all the components required for protein translation, including ribosomes derived from human cells, translation initiation and elongation factors, and tRNA. Compared to traditional cell-based experiments, this product provides a rapid method for producing proteins and evaluating mRNA constructs, including those designed for vaccine development applications.

Preparation of FLuc mRNA with different 3 UTR sequences.Various 3′ UTR sequences from human α-globin, human β-globin, human amino-terminal enhancer of split (AES) gene, and hepatitis virus-derived woodchuck post-transcriptional regulatory element (WPRE) were each introduced downstream of the FLuc gene. The 5 UTR, FLuc sequence, and Poly(A) sequence remained the same for each DNA template. The templates were transcribed using Takara IVTpro T7 mRNA Synthesis Kit (Cat. #6144) and capped with CleanCap Reagent AG (3′ OMe) (TriLink, SKU: N-7413).

Temperature for maximum protein expression with the Human Cell-Free Protein Expression Master Mix for mRNA is protein-dependent.Two µg of capped and uncapped mRNA coding for eIF4G, FLuc, and ZsGreen were introduced to the Human Cell-Free Protein Expression Master Mix for mRNA and incubated at 27, 32, and 37 °C for 3 hours. High expression of the target proteins was only observed when capped mRNAs were introduced, as confirmed by SDS-PAGE (eIF4G & FLuc), where red arrows point to the desired protein band; chemiluminescence measurement (FLuc); or fluorescence measurement (ZsGreen). Temperature for maximum protein expression was protein-dependent. eIF4G reached maximum expression at 27 or 32 °C; FLuc at 27 °C; and ZsGreen at 32 °C. As supported by these experiments, the optimal reaction temperature for this cell-free system depends on the protein being expressed. mRNAs were prepared using the Cloning Kit for mRNA Template (BspQ I) (Cat. # 6133) and Takara IVTpro T7 mRNA Synthesis Kit (Cat. # 6144) and capped with CleanCap Reagent AG (3′ OMe) (TriLink, SKU: N-7413).

Comparing FLuc protein expression between Human Cell-Free Protein Expression Master Mix for mRNA and HEK293T cells.Two μg of each capped FLuc mRNA construct were added to the Human Cell-Free Protein Expression Master Mix for mRNA and incubated at 27 °C for two hours (Panel A). mRNA (0.5 μg) was transfected into HEK293T and cultured for 24 hours (Panel B). FLuc protein activity was measured by chemiluminescence. A high correlation was found between the FLuc protein expression between cell-free and cell-expressed systems (Panel C, R2= 0.87). This suggests that Human Cell-Free Protein Expression Master Mix for mRNA, can be a simple and rapid way to evaluate and screen mRNA constructs.

Human Cell-Free Protein Expression Master Mix for mRNA successfully produces proteins of varying molecular weights.Capped and uncapped mRNA coding for eIF4G (170 kDa), FLuc (61 kDa), and ZsGreen (26 kDa) were introduced to the Human Cell-Free Protein Expression Master Mix for mRNA at three different input amounts (1, 2, or 4 µg) and incubated for three hours at 32 °C. High expression of the target proteins was only observed when capped mRNAs were introduced, as confirmed by SDS-PAGE (eIF4G & FLuc), where red arrows point to the desired protein band; chemiluminescence measurement (FLuc); or fluorescence measurement (ZsGreen). This indicates that mRNA expression is cap-dependent for this cell-free expression system. For these experiments, protein expressions were maximized with an mRNA input amount of 2 µg. mRNAs were prepared using the Cloning Kit for mRNA Template (BspQ I) (Cat. # 6133) and Takara IVTpro T7 mRNA Synthesis Kit (Cat. # 6144) and capped with CleanCap Reagent AG (3′ OMe) (TriLink, SKU: N-7413).

Time to maximum protein expression with the Human Cell-Free Protein Expression Master Mix for mRNA is protein-dependent.Two µg of capped and uncapped mRNA coding for eIF4G, FLuc, and ZsGreen were introduced to the Human Cell-Free Protein Expression Master Mix for mRNA and incubated at 32 °C for 0, 1, 2, 3, and 5 hours. High expression of the target proteins was only observed when capped mRNAs were introduced, as confirmed by SDS-PAGE (eIF4G & FLuc), where red arrows point to the desired protein band; chemiluminescence measurement (FLuc); or fluorescence measurement (ZsGreen). Maximum protein expression was protein-dependent. eIF4G reached maximum expression at 3–5 hours; FLuc at 2 hours; and ZsGreen at 5 hours. As supported by these experiments, the optimal reaction time for this cell-free system depends on the protein being expressed. mRNAs were prepared using the Cloning Kit for mRNA Template (BspQ I) (Cat. # 6133) and Takara IVTpro T7 mRNA Synthesis Kit (Cat. # 6144) and capped with CleanCap Reagent AG (3′ OMe) (TriLink, SKU: N-7413).