Expression of Met-(-1) angiogenin in Escherichia coli: conversion to the authentic less than 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 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 less than 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.
Shapiro
R
R
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Harper
J W
JW
Fox
E A
EA
Jansen
H W
HW
Hein
F
F
Uhlmann
E
E
D000595:Amino Acid Sequence; D043925:Angiogenesis Inducing Agents / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005813:Genes, Synthetic; D006133:Growth Substances / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D011506:Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012259:Ribonuclease, Pancreatic
D043925:Angiogenesis Inducing Agents; D003062:Codon; D006133:Growth Substances; D009363:Neoplasm Proteins; D011506:Proteins; D011994:Recombinant Proteins; C058968:angiogenin, methionyl-(-1)-; C046366:angiogenin; D012259:Ribonuclease, Pancreatic