The α-1 antitrypsin Z-mutant protein (ATZ) is the primary cause of α-1 antitrypsin deficiency (AATD). Studying the ubiquitination modification and degradation of ATZ protein is importance for developing treatments for AATD. STUB1 is an important E3 ubiquitin ligase that regulates ubiquitination modification of various proteins. However, whether STUB1 in involved in the ubiquitination modification of ATZ has not been fully elucidated. In this study, the ATZ and STUB1 coding genes were first cloned into the pET28a plasmid, constructing 2 protein expression plasmids. The recombinant plasmids were then transferred into the Escherichia coli for expression. With the optimization of induction temperature and IPTG dosage, the recombinant proteins were successfully expressed. The target proteins were then efficiently purified from cell lysates using metal-chelating affinity chromatography, and the accuracy of the amino acid sequence was verified through protein mass spectrometry analysis. Using the purified ATZ and STUB1, we established an in vitro ubiquitination reaction system. Experimental results showed that, in the presence of ATP, E1 ubiquitin-activating enzyme, and E2 ubiquitin-conjugating enzyme, STUB1 catalyzed the ubiquitination modification of ATZ. This study provides a method for obtaining the ATZ protein in vitro, elucidates the mechanism of STUB1 mediating ATZ ubiquitination, thereby advancing our understanding of the intracellular degradation mechanism of the α-1 antitrypsin Z-mutant.