增强蛋白质的稳定性在蛋白质工程领域中至关重要。使用肽标记系统的蛋白质自环化已成为增强靶蛋白质热稳定性的有效策略。在这项研究中,我们利用了一个新的肽标记系统,ReverseTag/ReverseCatcher,利用分子内酯键的形成。最初,我们使用GFP作为模型来验证ReverseTag/ReverseCatcher介导的环化在提高蛋白质热稳定性方面的可行性。在100°C孵育30分钟后,环化的GFP(cGFP)保留了其相对荧光的30%,而GFP和线性GFP(lGFP)在5分钟内完全失去其荧光。此外,我们将这种方法应用于外切菊粉酶(EXINU),产生一个名为环化EXINU(cEXINU)的变体。cEXINU的T50和t1/2值表现出10°C和10分钟的显着增强,分别,与EXINU相比。此外,后环化,EXINU具有5至10的宽操作pH范围,具有持续的催化活性,和cEXINU在pH5和9下维持960分钟的半衰期。进行分子动力学模拟以阐明环化后EXINU的热稳定性和pH稳定性增强的潜在机制。这项研究强调,环化实质上增强了高稳定蛋白GFP和低稳定蛋白EXINU的稳定性,由ReverseTag/ReverseCatcher标记系统介导。ReverseTag/ReverseCatcher标记系统被证明是一种有效的共轭方法,在提高热稳定性方面具有潜在的应用,pH值稳健性,和蛋白质工程的其他领域。
Enhancing protein stability is pivotal in the field of protein engineering. Protein self-
cyclization using peptide a tagging system has emerged as an effective strategy for augmenting the thermostability of target proteins. In this study, we utilized a novel peptide tagging system, ReverseTag/ReverseCatcher, which leverages intramolecular ester bond formation. Initially, we employed GFP as a model to validate the feasibility of
cyclization mediated by ReverseTag/ReverseCatcher in improving the protein thermostability. Cyclized GFP (cGFP) retained 30 % of its relative fluorescence after a 30-min incubation at 100 °C, while both GFP and linear GFP (lGFP) completely lost their fluorescence within 5 min. Additionally, we applied this method to exo-inulinase (EXINU), resulting in a variant named cyclized EXINU (cEXINU). The T50 and t1/2 values of cEXINU exhibited significant enhancements of 10 °C and 10 min, respectively, compared to EXINU. Furthermore, post-
cyclization, EXINU demonstrated a broad operational pH range from 5 to 10 with sustained catalytic activity, and cEXINU maintained a half-life of 960 min at pH 5 and 9. Molecular dynamics simulations were conducted to elucidate the mechanisms underlying the enhanced thermostability and pH robustness of EXINU following
cyclization. This study highlights that
cyclization substanitially enhances the stability of both highly stable protein GFP and low-stable protein EXINU, mediated by the ReverseTag/ReverseCatcher tagging system. The ReverseTag/ReverseCatcher tagging system proves to be a potent conjugation method, with potential applications in improving thermostability, pH robustness, and other areas of protein engineering.