mRNA encoding Enhanced green fluorescent protein
eGFP mRNA encodes Enhanced Green Fluorescent Protein (eGFP), the most widely used fluorescent reporter in cell biology and molecular research. eGFP is a humanized variant of wild-type GFP originally isolated from the jellyfish Aequorea victoria, engineered to overcome the limitations of the native protein. Two key point mutations, F64L and S65T, dramatically improve performance in mammalian systems: F64L enhances chromophore maturation at 37°C, while S65T simplifies the excitation spectrum to a single peak at ~488 nm and increases brightness approximately 35-fold over wild-type GFP. The coding sequence was further optimized with over 190 silent nucleotide substitutions to reflect preferred human codon usage, yielding an additional ~4-fold increase in mammalian expression. The resulting protein fluoresces bright green (excitation 488 nm, emission 507 nm), requires no exogenous cofactors, and is compatible with standard fluorescein filter. The mRNA construct includes a 5' Cap1 structure and poly(A) tail to support efficient translation in mammalian cell-free and cellular expression systems.
Applications:
1) Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., & Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science, 263(5148), 802–805
2) Cormack, B. P., Valdivia, R. H., & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene, 173(1), 33–38
3) Yang, T. T., Cheng, L., & Kain, S. R. (1996). Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Research, 24(22), 4592–4593
4) Tsien, R. Y. (1998). The green fluorescent protein. Annual Review of Biochemistry, 67, 509–544
5) Zeng, C., Hou, X., Yan, J., Zhang, C., Li, W., Zhao, W., Du, S., & Dong, Y. (2020). Leveraging mRNA sequences to express SARS-CoV-2 antigens in vivo. Cold Spring Harbor Laboratory
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PRIME is Cisterna's core RNA production platform, capable of synthesizing all RNA types including mRNA, tRNA, siRNA, sgRNA, aptamers, and more. The PRIME platform is engineered to drastically reduce product-related impurities such as dsRNA, short-truncated products, abortive transcripts, and DNA:RNA hybrids, all commonly generated in standard in vitro transcription. The result is consistently high-quality RNAs that are fully functional and well-suited for most downstream applications such as X-ray crystallography, Cryo-EM, tRNA aminoacylation and protein expression.
Both PRIME and SPECC reflect Cisterna’s commitment to producing high-quality RNA, but they are designed to serve different needs. PRIME is Cisterna’s standard production platform and is applicable to all RNA types. It delivers high-purity RNA suitable for a wide range of applications, from structural and biochemical studies to robust protein expression. For most research applications, PRIME offers excellent performance and exceptional value at an affordable price point. SPECC (SPEcific Capture and Cleavage) is Cisterna’s flagship purification system and is currently available for mRNA only. It takes purity a step further by selectively isolating full-length mRNA transcripts, enabling near-HPLC-level purity. SPECC is the ideal choice when the highest possible mRNA quality is desired, such as in rigorous quantitative studies, high-dose applications, or therapeutic development.