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

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 bacter...

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Published inAnalytical 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
LanguageEnglish
Published San Diego, CA Elsevier Inc 1988
Elsevier
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Summary: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.
Bibliography:ObjectType-Article-2
SourceType-Scholarly Journals-1
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content type line 23
ISSN:0003-2697
1096-0309
DOI:10.1016/0003-2697(88)90569-6