The conventional peptide substrates of SARS-CoV-2 main protease(Mpro)are frequently associated with high cost,unstable kinetics,and multistep synthesis.Hence,there is an urgent need to design affordable and stable Mpro substrates for pharmacological research.Herein,we designed a functional Mpro substrate based on a dimerization-dependent red fluorescent protein(ddRFP)for the evaluation of Mpro inhibitors in vitro.The codon-optimized DNA fragment encoding RFP-A1 domain,a polypeptide linker containing Mpro cleavage sequence(AVLQS),and the RFP-B1 domain was subcloned into the pET-28a vector.After transformation into Escherichia coli Rosetta(DE3)cells,the kanamycin resistant transformants were selected.Using a low temperature induction strategy,most of the target proteins(ddRFP-M)presented in the supernatant fractions were collected and purified by a HisTrapTM chelating column.Subsequently,the inhibition of Mpro by ensitrelvir and baicalein was assessed using ddRFP-M assay,and the biochemical properties of ddRFP-M substrate were analyzed.Our results showed that the fluorogenic substrate ddRFP-M was successfully prepared from E.coli cells,and this biosensor exhibited the expected specificity,sensitivity,and reliability.In conclusion,the production of the fluorogenic substrate ddRFP-M provides an expedient avenue for the assessment of Mpro inhibitors in vitro.
关键词
新冠病毒/主蛋白酶/荧光底物/二聚化红色荧光蛋白/恩赛特韦/黄芩素
Key words
SARS-CoV-2/main protease/fluorogenic substrate/dimerization-dependent red fluorescent protein/ensitrelvir/baicalein
维普
万方数据