Expression And Purification Of QueF-Like Enzyme With ?-tail

Archaea is also called archaeobacteria.They share many similarities with prokaryotes and some features of eukaryotes.Archaea consist of two groups,Euryarchaeota and Crenarchaeota.Archaeosine(G+)is a highly modified nucleoside found in the tRNA of archaeans.Studies have shown that the Quef-like enzyme involved in the synthesis of Archaeosine(G+)in the archaea have homology with the QueF enzyme of the bacteria.QueF gene was cloned from bacteria,and the nitrile reductase activity has been found in the catalytic activity of QueF.The nitrile reductase has a good application prospect.Therefore,it is of great significance to study the activity of Quef-like enzymes from archaea.According to reports,the fusion protein with ?-tail can be expressed in bacteria,and when mixed with the ?-Sheet Fibrillizing peptide,they can be coinduced and assembled into nanofiber structure,and in this nanofiber structure the catalytic efficiency and stability of the enzyme can be improved.Therefore,the Quef-like gene of Pyrobaculum calidifontis was selected in this study,and the coding sequence of(3-tail was added to the c-terminal of the Quef-like gene,and the complete sequence was synthesized by Bao Sheng Wu company in dalian.In this study,the whole synthetic sequence was removed from pUC-19 and inserted into the vector pET-28a through gene recombination technology,and the recombinant expression vector pET-28a-Quef-like-?-tail was reconstructed,and then the Quef-like protein with ?-tail was expressed in Escherichia coli and the expressed protein has been purified.SDS-PAGE indicated that the target protein was expressed successfully.This study is the first step research work for the subsequent assembly of the QueF-Like enzyme nanofibers.