Browse > Article
http://dx.doi.org/10.4014/jmb.0905.05055

Design and Expression of Recombinant Antihypertensive Peptide Multimer Gene in Escherichia coli BL21  

Rao, Shengqi (State Key Laboratory of Food Science and Technology, Jiangnan University)
Su, Yujie (State Key Laboratory of Food Science and Technology, Jiangnan University)
Li, Junhua (State Key Laboratory of Food Science and Technology, Jiangnan University)
Xu, Zhenzhen (School of Food Science and Technology, Jiangnan University)
Yang, Yanjun (State Key Laboratory of Food Science and Technology, Jiangnan University)
Publication Information
Journal of Microbiology and Biotechnology / v.19, no.12, 2009 , pp. 1620-1627 More about this Journal
Abstract
The design and expression of an antihypertensive peptide multimer (AHPM), a common precursor of 11 kinds of antihypertensive peptides (AHPs) tandemly linked up according to the restriction sites of gastrointestinal proteases, were explored. The DNA fragment encoding the AHPM was chemically synthesized and cloned into expression vector pGEX-3X. After an optimum induction with IPTG, the recombinant AHPM fused with glutathione S-transferase (GST-AHPM) was expressed mostly as inclusion body in Escherichia coli BL21 and reached the maximal production, 35% of total intracellular protein. The inclusion body was washed, dissolved, and purified by cation-exchange chromatography under denaturing conditions, followed by refolding together with size-exclusion chromatography and gradual dialysis. The resulting yield of the soluble GSTAHPM (34 kDa) with a purity of 95% reached 399 mg/l culture. The release of high active fragments from the AHPM was confirmed by the simulated gastrointestinal digestion. The results suggest that the design strategy and production method of the AHPM will be useful to obtain a large quantity of recombinant AHPs at a low cost.
Keywords
Antihypertensive peptide; antihypertensive peptide multimer; GST fusion protein; expression; design;
Citations & Related Records
Times Cited By KSCI : 1  (Citation Analysis)
Times Cited By Web Of Science : 4  (Related Records In Web of Science)
연도 인용수 순위
1 Cushman, D. W. and H. S. Cheung. 1971. Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem. Pharmacol. 20: 1637-1648   DOI   ScienceOn
2 Fujita, H., K. Yokoyama, and M. Yoshikawa. 2000. Classification and antihypertensive activity of angiotensin I-converting enzyme inhibitory peptides derived from food proteins. J. Food Sci. 65: 564-569   DOI   ScienceOn
3 Fujita, H., K. Yokoyama, and M. Yoshikawa. 2000. Classification and antihypertensive activity of angiotensin I-converting enzyme inhibitory peptides derived from food proteins. J. Food Sci. 65: 564-569   DOI   ScienceOn
4 Glasser, S. P. 2001. Hypertension syndrome and cardiovascular events. Postgrad. Med. 110: 29-36   PUBMED   ScienceOn
5 Israaili, Z. H. and W. D. Hall. 1992. Cough and angioneurotic edema associated with angiotensin converting enzyme inhibitor therapy: A review of the literature and pathophysiology. Ann. Intern. Med. 117: 234-242   DOI   PUBMED   ScienceOn
6 Jeong, D. W., D. S. Shin, C. W. Ahn, I. S. Song, and H. J. Lee. 2007. Expression of antihypertensive peptide, His-His-Leu, as tandem repeats in Escherichia coli. J. Microbiol. Biotechnol. 17: 952-959   과학기술학회마을   PUBMED   ScienceOn
7 Li, G. H., G. W. Le, and Y. H. Shi. 2004. Shrestha, angiotensin I-converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects. Nutr. Res. 24: 469-486   ScienceOn
8 Lv, G. S., G. C. Huo, and X. Y. Fu. 2003. Expression of milkderived antihypertensive peptide in Escherichia coli. J. Dairy Sci. 86: 1927-1931   DOI   ScienceOn
9 Megias, C., J. Pedroche, M. D. M. Yust, M. Alaiz, J. Giron-Calle, F. Millan, and J. Vioque. 2006. Affinity purification of angiotensin converting enzyme inhibitory peptides using immobilized ACE. J. Agric. Food Chem. 54: 7120-7124   DOI   ScienceOn
10 Murray, B. A. and R. J. FitzGerald. 2007. Angiotensin converting enzyme inhibitory peptides derived from food proteins: Biochemistry, bioactivity and production. Curr. Pharm. Des. 13: 773-791   DOI   ScienceOn
11 Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, U.S.A
12 Yamada, Y., N. Matoba, H. Usui, K. Onishi, and M. Yoshikawa. 2002. Design of a highly potent anti-hypertensive peptide based on ovokinin(2-7). Biosci. Biotech. Biochem. 66: 1213-1217   DOI   ScienceOn
13 Fujita, H. and M. Yoshikawa. 1999. LKPNM: A prodrugtype ACE-inhibitory peptide derived from fish protein. Immunopharmacology 44: 123-127   DOI   ScienceOn
14 Marczak, E. D., H. Usui, H. Fujita, Y. J. Yang, M. Yokoo, A. W. Lipkowski, and M. Yoshikawa. 2003. New antihypertensive peptides isolated from rapeseed. Peptides 24: 791-798   DOI   ScienceOn
15 Yamamoto, N., M. Maeno, and T. Takano. 1999. Purification and characterization of an antihypertensive peptide from a yogurt-like product fermented by Lactobacillus helveticus CPN4. J. Dairy Sci. 82: 1388-1393   DOI   ScienceOn
16 Studier, F. W. and B. A. Moffatt. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189: 113-130   DOI   PUBMED
17 FitzGerald, R. J., B. A. Murray, and D. J. Walsh. 2004. Hypotensive peptides from milk proteins. J. Nutr. 134: 980-988
18 Sato, M., T. Hosokawa, T. Yamaguchi, T. Nakano, K. Muramoto, T. Kahara, K. Funayama, A. Kobayashi, and T. Nakano. 2002. Angiotensin I-converting enzyme inhibitory peptides derived from wakame (Undaria pinnatifida) and their antihypertensive effect in spontaneously hypertensive rats. J. Agric. Food Chem. 50: 6245-6252   DOI   ScienceOn
19 Makrides, S. C. 1996. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 60: 512-538   PUBMED   ScienceOn
20 Yamamoto, S., I. Toida, and K. Iwai. 1980. Re-examination of the spectrophotometric assay for serum angiotensin-converting enzyme. Nippon Kyobu Shippei Kaishi 18: 297-303
21 Bettadapura, J., K. K. Menon, S. Moritz, J. Liu, and C. C. Bernard. 1998. Expression, purification, and encephalitogenicity of recombinant human myelin oligodendrocyte glycoprotein. J. Neurochem. 70: 1593-1599   DOI   ScienceOn
22 Liu, D., H. Y. Sun, L. J. Zhang, and S. M. Li. 2007. High-level expression of milk-derived antihypertensive peptide in Escherichia coli and its bioactivity. J. Agric. Food Chem. 55: 5109-5112   DOI   ScienceOn
23 Smith, D. B. and K. S. Johnson. 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67: 31-40   DOI   ScienceOn
24 Tauzin, J., L. Miclo, and J. L. Gaillard. 2002. Angiotensin-Iconverting enzyme inhibitory peptides from tryptic hydrolysate of bovine alpha(S2)-casein. FEBS Lett. 531: 369-374   DOI   ScienceOn
25 Megias, C., M. D. M. Yust, J. Pedroche, H. Lquari, J. Giron-Calle, M. Alaiz, F. Millan, and J. Vioque. 2004. Purification of an ACE inhibitory peptide after hydrolysis of sunflower (Helianthus annuus L.) protein isolates. J. Agric. Food Chem. 52: 1928-1932   DOI   ScienceOn
26 Byun, H. G. and S. K. Kim. 2001. Purification and characterization of angiotensin I converting enzyme (ACE) inhibitory peptides from Alaska pollack (Theragra chalcogramma) skin. Process Biochem. 36: 1155-1162   DOI   ScienceOn
27 Chen, G. W., J. S. Tsai, and B. S. Pan. 2007. Purification of angiotensin I-converting enzyme inhibitory peptides and antihypertensive effect of milk produced by protease-facilitated lactic fermentation. Int. Dairy J. 17: 641-647   DOI   ScienceOn
28 Nakamura, Y., N. Yamamoto, K. Sakai, A. Okubo, S. Yamazaki, and T. Takano. 1995. Purification and characterization of angiotensin I-converting enzyme inhibitors from sour milk. J. Dairy Sci. 78: 777-783   DOI   ScienceOn
29 Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254   DOI   PUBMED   ScienceOn
30 Onishi, K., N. Matoba, Y. Yamada, N. Doyama, N. Maruyama, S. Utsumi, and M. Yoshikawa. 2004. Optimal designing of betaconglycinin to genetically incorporate RPLKPW, a potent antihypertensive peptide. Peptides 25: 37-43   DOI   PUBMED   ScienceOn
31 Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685   DOI   PUBMED   ScienceOn
32 Park, C. J., J. H. Lee, S. S. Hong, H. S. Lee, and S. C. Kim. 1998. High-level expression of the angiotensin-converting-enzymeinhibiting peptide, YG-1, as tandem multimers in Escherichia coli. Appl. Microbiol. Biotechnol. 50: 71-76   DOI   ScienceOn
33 Wang, J. P., J. E. Hu, J. Z. Cui, X. F. Bai, Y. G. Du, Y. Miyaguchi, and B. C. Lin. 2008. Purification and identification of a ACE inhibitory peptide from oyster proteins hydrolysate and the anti hypertensive effect of hydrolysate in spontaneously hypertensive rats. Food Chem. 111: 302-308   DOI   ScienceOn
34 Alting, A. C., R. J. Meijer, and E. C. van Beresteijn. 1997. Incomplete elimination of the ABBOS epitope of bovine serum albumin under simulated gastrointestinal conditions of infants. Diabetes Care 20: 875-880   DOI   ScienceOn