Cyclization of the polypeptide backbone has proven to be a powerful strategy for enhancing protein stability for fundamental research and pharmaceutical application. The use of such an approach is restricted by how well a targeted polypeptide can be efficiently ligated. Recently, an Asx-specific peptide ligase identified from a tropical cyclotide-producing plant and named butelase 1 exhibited excellent cyclization kinetics that cannot be matched by other known ligases, including intein, PATG, PCY1, and sortase A. In this work, we aimed to examine whether butelase 1 facilitated protein conformational stability for structural investigation. First, we successfully expressed recombinant butelase 1 (rBTase) in the yeast Pichia pastoris. Next, rBTase was shown to be highly efficient in the cyclization of the p53-binding domain (N-terminal domain) of murine double minute X (N-MdmX), an important target for designing anticancer drugs. The cyclized N-MdmX (cMdmX) exhibited increased conformational stability and improved interaction with the ligand compared with those of noncyclized N-MdmX. Importantly, the thermal melting process was completely reversible, contrary to noncyclized N-MdmX, and the melting temperature (T m ) of cMdmX was increased to 47 from 43 °C. This stable conformation of cMdmX was further confirmed by 15 N− 1 H heteronuclear single-quantum coherence nuclear magnetic resonance (NMR) spectroscopy. The complex of cMdmX and the ligand was tested for protein crystallization, and several promising findings were revealed. Therefore, our work not only provides a recombinant version of butelase 1 but also suggests a conventional approach for preparing stable protein samples for both protein crystallization and NMR structural investigation.
The readthrough of premature termination codons (PTCs) is a promising strategy for curing PTC‐causing diseases. In cancers, the p53 anti‐tumor activity is often disabled by forming premature terminated p53 protein (p53PTC). Currently, there are lack of p53PTC‐rescuing drugs. Herein we designed a feasible strategy for identifying p53PTC‐rescuing compounds using protein biosynthesis machinery in E. coli cells and the lung cancer H1299 cells both harboring p53PTC‐GFP fusion protein expression cassettes. Rescued p53PTC protein enabled a GFP‐tag for fluorescence spectroscopic measurements. Our data revealed that the aminoglycoside G418 not only efficiently rescued p53PTC in H1299 cells, but also synergistically enhanced the efficacy of antitumor drug doxorubicin. Our work gave an insight into the discovery of p53PTC‐rescuing drugs for cancer therapy.
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