Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45.
By chemoenzymatic synthesis the gene for a (Leu27) analogue of human growth hormone releasing hormone-Gly45 [(Leu27)GHRH-Gly45] was constructed, cloned and expressed in Escherichia coli as a fusion protein with beta-galactosidase under the control of the lac promoter and operator. Upon induction with isopropyl-D-thio-beta-galactopyranoside the fusion protein accumulated to a yield of 15-20% of the total cellular protein. After cyanogen bromide cleavage of the fusion protein the precursor peptide (Leu27)hGHRH-Gly45 was separated by extraction and purified by ion exchange and h.p.l.c.-RP18 chromatography. The purified peptide was analysed by sequencing, isoelectric focusing, amino acid analysis and amino acid analysis after V8 protease digestion. The carboxy-terminal glycine was subsequently amidated by PAM (peptidylglycine-alpha-amidating-monooxygenase), an enzyme which was isolated and characterized from fresh bovine pituitaries. Correct amidation of the penultimate amino acid, leucine, was verified by peptide sequencing with an authentic leucine amide reference.
Engels
J W
JW
Universität Frankfurt, Institut für Organische Chemie, Frankfurt/Main, FRG.
Glauder
J
J
Müllner
H
H
Tripier
D
D
Uhlmann
E
E
Wetekam
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D013007:Growth Hormone-Releasing Hormone / Q000031:analogs & derivatives* / Q000235:genetics / Q000302:isolation & purification; D006899:Mixed Function Oxygenases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes; D000587:Oxidoreductases Acting on CH-NH Group Donors; D015202:Protein Engineering
D009097:Multienzyme Complexes; C050990:somatotropin releasing hormone (1-45), 27-Leu-45-Gly-; D013007:Growth Hormone-Releasing Hormone; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D000587:Oxidoreductases Acting on CH-NH Group Donors