Tandem multimer expression of angiotensin I-converting enzyme inhibitory peptide in Escherichia coli.
It is common for small tandem peptide multimer genes to be indirectly inserted into expression vectors and fused with a protein tag. In this study, a multimer of the tandem angiotensin I-converting enzyme inhibitory peptide (ACE-IP) gene was directly transferred to a commercially available vector and the designed gene was expressed as a repeated peptide in Escherichia coli BL21(DE3)pLysS. The process further developed in our study was the construction of six-repeated ACE-IP synthetic genes and their direct insertion. Protein expression in inclusion bodies was confirmed by SDS-PAGE and Western blot. Acid hydrolysis of inclusion bodies produced single-unit peptides through cleavage of the aspartyl-prolyl bonds. This cleaved recombinant peptide (rACE-IP) was purified using immuno-affinity chromatography followed by reversed phase-HPLC. 105-115 mg of the lyophilized recombinant peptide was obtained from 1 L E. coli culture. In vitro biological activity of rACE-IP was indistinguishable from that of the natural peptide produced by hydrolysis in artificial gastric juice or by acidic hydrolysis. The rACE-IP prepared by recombinant DNA technology and solid-phase synthesis methods showed a similar IC(50). This strategy could be used for the expression of important peptides, which have N-terminal proline (P) and C-terminal aspartic acid residues (D) for commercial applications, e.g. functional foods and drinks.
Fida
Hasan M
HM
Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Faculty of Engineering, Kobe University, Kobe, Japan.
Kumada
Yoichi
Y
Terashima
Masaaki
M
Katsuda
Tomohisa
T
Katoh
Shigeo
S
D000806:Angiotensin-Converting Enzyme Inhibitors / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000502:physiology*; D010455:Peptides / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism*
D000806:Angiotensin-Converting Enzyme Inhibitors; D010455:Peptides; D011994:Recombinant Proteins