high efficient green fluorescent protein (GFP) using novel molecular engineering approaches - beyond the biology

Abstract

Introduction: Green fluorescent proteins (GFPs) and GFP-like proteins from marine organisms, especially those from the jellyfish Aequorea victoria, called avGFP, have made significant contributions to cell biology. Because of their unique protein sequence, they have light-absorbing chromophores. avGFP attracted much attention after its cloning in 1992. Since then, many researchers have focused on identifying and improving fluorescent proteins with new qualities and enhanced properties for direct observation of dynamic processes in living cells and tissues. Molecular engineering of GFP as a powerful toolkit helps us to produce brighter fluorescent light at a specific wavelength or enhance photostability and photoactivation properties. Also, in some variants, chromophores form rapidly and Effectively. moreover, some engineered versions sense physiological signals within cells, like concentrations of free ions. actually, they act as biosensors. Methods: In this review, we studied more than 40 articles from 1995 to 2022 from valid databases. clearly, we describe the structure and molecular characterization of GFP at first and then focus on the engineered versions, their novel properties, and developments. Results: GFP as a popular protein molecule has been noticed in recent years and new desirable features have been added to it with various engineering methods. The evolution of cell biology was realized by using GFP as a reporter gene and monitoring the gene expression. In addition, other applications like using as intracellular markers, Subcellular localization, cell trafficking, biosensors, protein-protein interactions and photobleaching (to investigate protein dynamics in living cells) are remarkable. However, the wild type of GFP has limitations that must be overcome by protein engineering techniques or site-directed mutagenesis for optimal use. Conclusion: In recent years GFPs and GFP-like proteins have been the subject of Many studies and so far many efforts have been made to overcome their structural and functional limitations. By using protein engineering techniques, limitations such as weak folding at 37°C, inappropriate absorption peaks, low photostability or low speed of chromophore formation have been