High-throughput optimization of peptide-linker for fusing function protein with GFP

Fusion proteins are pivotal in bioengineering, with applications in purification , delivery, and imaging. However, the development of specialized peptide linkers tailored for target fusion proteins remains an unmet challenge. In this study, we demonstrate the optimization of fusing a functional protein with green fluorescent protein (GFP) through the screening of peptide linker sequences. Using seamless cloning methodology, a nanobody protein was fused to the N-terminus of GFP via a randomized 18-amino acid peptide linker library. Initial screening of fusion protein clones was conducted on solid plates to identify those expressing robust GFP fluorescence. A total of 153 clones with unique linker sequences were identified using Sanger sequencing . A wide range of normalized fluorescence signals was observed, revealing significant variability in linker performance. Among the screened linkers, one exhibited high fluorescence activity, outperforming commonly used flexible and rigid linkers. This finding underscores the necessity of optimize linker sequences for specific fusion proteins. Furthermore, the results demonstrated that the screened linker is compatible with diverse N-terminal proteins while maintaining GFP functionality. Additionally, to investigate the effect of linker on the function of target protein, we determined the reverse transcription efficiency of the murine leukemia virus reverse transcriptase (MLV-RT) in the fusion proteins by a two-step RT-qPCR method. In conclusion, this study presents an efficient optimization of peptide linkers, offering a novel methodology for the engineering and application of specialized linkers for fusion proteins. • A peptide linker library was constructed for high-throughput screening. • 153 fusion proteins containing unique linker sequences were successfully expressed in E. coli BL21. • Among the screened linkers, one exhibited high fluorescence activity, outperforming commonly used flexible and rigid linkers. • The compatibility of the screened peptide linkers for fusion with different functional proteins was further validated.