Expression of Met-(−1) angiogenin in Escherichia coli: Conversion to the authentic <Glu-1 protein

• Published in: Analytical biochemistry Vol. 175; no. 2; pp. 450 - 461
• Main Authors: Shapiro, Robert, Harper, J.Wade, Fox, Edward A., Jansen, Hans-Willi, Hein, Friedrich, Uhlmann, Eugen
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 1988; Elsevier
• Subjects: aminopeptidase; angiogenesis; angiogenin; Biological and medical sciences; Biotechnology; Escherichia coli; Fundamental and applied biological sciences. Psychology; Gene expression; genes; Genetic engineering; Genetic technics; methionine; Methods. Procedures. Technologies; Molecular and cellular biology; Molecular cloning; Molecular genetics; mutagenesis; protein processing; trp promoter; mutagenesis; angiogenesis; protein processing; trp promoter; aminopeptidase; Human; Purification; Nucleotide sequence; Escherichia coli; Methionine; Recombinant DNA; Gene expression; Plasmid; Proteins; Aminoacid; Bacteria; Chemical composition; Genetic engineering; Molecular cloning; Escherichieae; Enterobacteriaceae
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(88)90569-6

A method for obtaining authentic human angiogenin utilizing an Escherichia coli recombinant expression system is described. A synthetic gene encoding angiogenin was placed into a vector for direct expression under the control of a modified E. coli trp promoter. The protein was produced by the bacteria in an insoluble form and purified to homogeneity by cation-exchange and reversed-phase HPLC following reduction/solubilization and reoxidation. The protein isolated was identified as Met-(−1) angiogenin by amino acid analysis and tryptic peptide mapping; the latter demonstrated that all three disulfide bonds had formed correctly. Both the enzymatic and angiogenic activities of the Met-(−1) protein were equivalent to those of native angiogenin. A Met-(−1) Leu-30 derivative of angiogenin was also isolated and found to be fully active. Conversion of Met-(−1) angiogenin to the authentic <Glu-1 protein was achieved by treatment with Aeromonas aminopeptidase under conditions in which the new N-terminal glutamine readily cyclizes nonenzymatically. This aminopeptidase treatment may have more general applicability for removal of undesirable N-terminal methionine residues from foreign proteins expressed in bacteria.