Preparation, and enzymatic activity analysis of an engineered capping enzyme

The Vaccinia capping enzyme (VCE) and the 2’-O-methyltransferase (VP39) are proteins encoded by the vaccinia virus genome, used for capping viral mRNA to form m⁷GpppN²Me mRNA (Cap1 mRNA). This capping structure is essential for protecting mRNA from degradation, facilitating pre-mRNA splicing and nuclear export, and enabling translation initiation by the eukaryotic initiation factor (eIF4E). Moreover, it helps the virus circumvent innate immune responses, thereby facilitating replication using host cell mechanisms. Currently, the enzymatic capping process employs VCE and VP39 in concert with pre-mRNA to synthesize Cap1 mRNA directly. This study introduces an engineered fusion capping enzyme , created by linking VCE and VP39 via a flexible (GGGGS)₃ linker(D1R-D12L-GS linker-VP39, DDGSV). The aim is to enhance the capping reaction while reducing raw material costs, process complexity, and impurities. The tertiary structure of DDGSV, predicted using AlphaFold2, aligns well with published structures of VCE and VP39, demonstrating no steric hindrance at the enzymatic active sites resulting from the fusion configuration. The expression vector pTolo-EX2-DDGSV was constructed and expressed in Escherichia coli BL21(DE3). The mRNA of the prepared capping enzymes exhibited good integrity on an agarose gel. The capping efficiency of the engineered enzyme DDGSV reached 80.19 % after 2 h of the capping reaction, matching the performance of commercial capping enzymes. Furthermore, the potential of RNA dot blotting for rapid detection of mRNA capping efficiency was explored; however, quantitative methods are also needed. Additionally, GFP mRNA prepared using DDGSV demonstrated high expression levels in HEK 293 T cells. These results indicate that the engineered enzyme can effectively cap Cap1 mRNA, providing a novel approach for mRNA vaccine development.