An expression vector (pSJyub-5) was constructed which contained five repeats of the "yeast ubiquitin gene" regulated by a heat-inducible lambda PL promoter. The vector, when expressed in Escherichia coli, produced a penta-ubiquitin of approximately 42 kDa. Purified penta-ubiquitin was found to be as active as the human mono-ubiquitin in the in vitro ATP/ubiquitin-dependent proteolytic assay of the reticulocyte lysate, indicating that the expressed gene product was recognized by the enzymes involved in the ATP/ubiquitin-dependent proteolytic pathway. The inability of penta-ubiquitin to act as a substrate in the pyrophosphate exchange reaction with the ubiquitin-activating enzyme E1 suggested that it had a carboxyl-terminal Asn, in agreement with the nucleotide sequence. In E. coli, the expressed penta-ubiquitin was processed correctly to mono-ubiquitin. The fidelity of processing in E. coli was confirmed by the following observations. The amino acid compositions of the processed mono-ubiquitin and human ubiquitin were similar. The 1H NMR spectrum of peaks representing amide hydrogens of the carboxyl-terminal Arg-74, Gly-75, and Gly-76 of the processed mono-ubiquitin was identical with that of human ubiquitin. The immunoreactivity of processed mono-ubiquitin and human ubiquitin against polyclonal antibodies that recognized epitope(s) only on the carboxyl terminus of ubiquitin were similar. The human and processed mono-ubiquitin were equally active in degrading 125I-bovine serum albumin in the ATP/ubiquitin-dependent in vitro proteolytic assay with reticulocyte lysates. In the pyrophosphate exchange assay with isolated ubiquitin activating enzyme E1, they were also equally reactive, confirming that the expressed and processed ubiquitin contained an intact carboxyl-terminal Gly-76. Purified penta-ubiquitin should prove to be a useful substrate for identifying and isolating processing enzyme(s) involved in the ATP/ubiquitin-dependent proteolytic pathway.