High-level expression of the angiotensin-converting-enzyme-inhibiting peptide, YG-1, as tandem multimers in Escherichia coli.
To produce a large quantity of the angiotensin-converting- enzyme(ACE)-inhibiting peptide YG-1, which consists of ten amino acids derived from yeast glyceraldehyde-3-phosphate dehydrogenase, a high-level expression was explored with tandem multimers of the YG-1 gene in Escherichia coli. The genes encoding YG-1 were tandemly multimerized to 9-mers, 18-mers and 27-mers, in which each of the repeating units in the tandem multimers was connected to the neighboring genes by a DNA linker encoding Pro-Gly-Arg for the cleavage of multimers by clostripain. The multimers were cloned into the expression vector pET-21b, and expressed in E. coli BL21(DE3) with isopropyl beta-D-thiogalactopyranoside induction. The expressed multimeric peptides encoded by the 9-mer, 18-mer and 27-mer accumulated intracellularly as inclusion bodies and comprised about 67%, 25% and 15% of the total proteins in E. coli respectively. The multimeric peptides expressed as inclusion bodies were cleaved with clostripain, and active monomers were purified to homogeneity by reversed-phase high-performance liquid chromatography. In total, 105 mg pure recombinant YG-1 was obtained from 11 E. coli culture harboring pETYG9 which contained the 9-mer of the YG-1 gene. The recombinant YG-1 was identical to the natural YG-1 in molecular mass, amino acid sequence and ACE-inhibiting activity.
Park
C J
CJ
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, Korea.
Lee
J H
JH
Hong
S S
SS
Lee
H S
HS
Kim
S C
SC
D000806:Angiotensin-Converting Enzyme Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry*; D002479:Inclusion Bodies / Q000737:chemistry; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000806:Angiotensin-Converting Enzyme Inhibitors; D001426:Bacterial Proteins; D010446:Peptide Fragments; D007544:Isopropyl Thiogalactoside; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003546:Cysteine Endopeptidases; C019913:clostripain