Structural and Functional Implications of Polyarginine Addition to Green Fluorescent Protein Expression in Saccharomyces cerevisiae INVSc1 [version 3; peer review: 4 not approved]

• Published in: F1000 research 2023, Vol.12, p.1
• Main Authors: Tandio Saputro, Shania Safera, Wahyunita, Khayu, Nurhasanah, Astutiati, Nugraha, Yudhi, Faizal, Irvan, Pambudi, Sabar, Wibowo, Syahputra, Pramono, Andri Pramesyanti
• Format: Article
• Language: English
• ISSN: 2046-1402; 2046-1402
• DOI: 10.12688/f1000research.123181.3

Green Fluorescent Protein (EGFP) is widely used as a reporter gene, aiding in protein recovery and transduction studies. In this study, EGFP was tagged with eleven arginine residues (PolyR) and six histidine residues (His-tag) for purification. The aim was to enhance the synthesis of EGFP-PolyR in Saccharomyces cerevisiae and evaluate the effects of polyarginine modification on protein stability and expression levels. The expression of EGFP and EGFP-PolyR in S. cerevisiae was assessed through fluorescence measurements and protein levels. Structural analyses were conducted using in silico tools to investigate changes in beta strands and helices, which were validated through Western blots. Results showed that EGFP-PolyR maintained similar fluorescence levels to EGFP, but with notable structural changes. EGFP-PolyR's final beta strand terminates at Ala228, compared to Gly229 in EGFP, affecting the beta sheet's stability. Structural modifications also included altered helix lengths, with a longer helix 10 and shorter helix 9 in EGFP-PolyR. These alterations, along with shifts in helix-helix interactions, contribute to destabilization. Additionally, EGFP-PolyR exhibited unique gamma coils absent in EGFP, further differentiating its structure. The structural changes led to decreased protein expression and solubility, as indicated by Western blot analysis, with EGFP-PolyR showing significantly lower expression levels. The findings suggest that EGFP-PolyR is prone to aggregation and misfolding, characteristics often associated with aggregation-prone proteins.In conclusion, the polyarginine modification significantly impacts the structural integrity, stability, and solubility of EGFP. While fluorescence is retained, these changes hinder protein detectability and purification, highlighting the importance of considering structural alterations when modifying reporter proteins for experimental use.