ABSTRACT Ubiquitination is involved in the regulation of numerous cellular functions. Research works in the ubiquitin realm rely heavily on ubiquitination assays in vitro and require large amounts of ubiquitin-activating enzyme (UBA1) and keep ATP supplies. However, UBA1 is hard to be obtained with large quantities using reported methods. We fused Escherichia coli adenylate kinase (adk) and mouse UBA1 and obtained fusion protein adk-mUBA1. The expression level of adk-mUBA1 increased about 8-fold compared with mUBA1 in an E. coli expression system, and adk-mUBA1 was easily purified to 90% purity via 2 purification steps. The purified adk-mUBA1 protein was functional for ubiquitination and could use ATP in addition to ADP as energy supply and had a higher catalytic activity than mUBA1 in cell lysis. adk-mUBA1 can be applied to preparing ubiquitin-modified substrates and kinds of ubiquitin chains in a chemical synthesis process and is a preferable application than mUBA1 in vitro ubiquitination.