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標題: 靈芝免疫調節蛋白融合抗氧化或降血脂胜肽於枯草桿菌之表現與活性測試
Fusion expression and function analysis of recombinant Ganoderma lucidum immunomodulatory protein LZ8 and antioxidative or hypocholesterolemic peptide
作者: 許廷豪
Hsu, Ting-Hao
關鍵字: 靈芝免疫調節蛋白;Bacillus subtilis;雙體結構;抗氧化胜肽降血脂胜肽融合蛋白;Ling-Zhi 8;dimer form;antioxidative peptide;hypocholesterolemic peptide;fusion protein
出版社: 食品暨應用生物科技學系所
引用: 蘇芳仙,2001。最佳σA啟動子及多重啟動子之構築及其於枯草桿菌中之表現。國立中興大學食品科學系碩士論文 羅秋梅,2005。建立地衣芽孢桿菌表現系統以應用於靈芝免疫調節蛋白之生產。國立中興大學食品暨應用生物科技學系碩士論文 林明義,2008。靈芝免疫調節蛋白質及其衍生功能性胜肽之設計、合成並於大腸 桿菌及枯草桿菌中表現純化。國立中興大學食品暨應用生物科技學系碩士 論文 <watching peptide.Pdf>. Akita, M., S. Sasaki, S. Matsuyama and S. Mizushima, 1990. Seca interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in escherichia coli. The Journal of biological chemistry, 265(14): 8164-8169. Chan, K.M. and E.A. Decker, 1994. Endogenous skeletal muscle antioxidants. Critical reviews in food science and nutrition, 34(4): 403-426. DOI 10.1080/10408399409527669. Chen, H.M., K. Muramoto, F. Yamauchi and K. Nokihara, 1996. Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein. Journal of Agricultural and Food Chemistry, 44(9): 2619-2623. Available from <Go to ISI>://WOS:A1996VH80700025. DOI 10.1021/jf950833m. Davis, C.E., B.M. Rifkind, H. Brenner and D.J. Gordon, 1990. A single cholesterol measurement underestimates the risk of coronary heart disease. An empirical example from the lipid research clinics mortality follow-up study. JAMA : the journal of the American Medical Association, 264(23): 3044-3046. Deuerling, E., A. Mogk, C. Richter, M. Purucker and W. Schumann, 1997. The ftsh gene of bacillus subtilis is involved in major cellular processes such as sporulation, stress adaptation and secretion. Molecular Microbiology, 23(5): 921-933. Available from <Go to ISI>://WOS:A1997WL76400007. DOI 10.1046/j.1365-2958.1997.2721636.x. Erdmann, K., N. Grosser, K. Schipporeit and H. Schroder, 2006. The ace inhibitory dipeptide met-tyr diminishes free radical formation in human endothelial cells via induction of heme oxygenase-1 and ferritin. The Journal of nutrition, 136(8): 2148-2152. Evans, J.L., I.D. Goldfine, B.A. Maddux and G.M. Grodsky, 2003. Are oxidative stress-activated signaling pathways mediators of insulin resistance and beta-cell dysfunction? Diabetes, 52(1): 1-8. Frankel, E.N. and A.S. Meyer, 2000. The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. Journal of the Science of Food and Agriculture, 80(13): 1925-1941. Available from <Go to ISI>://WOS:000089540300015. DOI 10.1002/1097-0010(200010)80:13<1925::aid-jsfa714>;2-4. Fu, L.L., Z.R. Xu, W.F. Li, J.B. Shuai, P. Lu and C.X. Hu, 2007. Protein secretion pathways in bacillus subtilis: Implication for optimization of heterologous protein secretion. Biotechnology advances, 25(1): 1-12. Available from <Go to ISI>://WOS:000243859800001. DOI 10.1016/j.biotechadv.2006.08.002. Gauthier, S.F., Y. Pouliot and D. Saint-Sauveur, 2006. Immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins. International Dairy Journal, 16(11): 1315-1323. DOI 10.1016/j.idairyj.2006.06.014. Gill, I., R. Lopez-Fandino, X. Jorba and E.N. Vulfson, 1996. Biologically active peptides and enzymatic approaches to their production. Enzyme and microbial technology, 18(3): 163-183. Haak-Frendscho, M., K. Kino, T. Sone and P. Jardieu, 1993. Ling zhi-8: A novel t cell mitogen induces cytokine production and upregulation of icam-1 expression. Cellular immunology, 150(1): 101-113. DOI 10.1006/cimm.1993.1182. Hartmann, R. and H. Meisel, 2007. Food-derived peptides with biological activity: From research to food applications. Current opinion in biotechnology, 18(2): 163-169. Available from DOI 10.1016/j.copbio.2007.01.013. Hoffmann, T., B. Troup, A. Szabo, C. Hungerer and D. Jahn, 1995. The anaerobic life of bacillus-subtilis - cloning of the genes encoding the respiratory nitrate reductase system. Fems Microbiology Letters, 131(2): 219-225. Available from <Go to ISI>://WOS:A1995RT10400017. DOI 10.1111/j.1574-6968.1995.tb07780.x. Hsu, H.C., C.I. Hsu, R.H. Lin, C.L. Kao and J.Y. Lin, 1997. Fip-vvo, a new fungal immunomodulatory protein isolated from volvariella volvacea. Biochemical Journal, 323: 557-565. Available from <Go to ISI>://WOS:A1997WV76200035. Huang, L., F. Sun, C. Liang, Y.X. He, R. Bao, L. Liu and C.Z. Zhou, 2009. Crystal structure of lz-8 from the medicinal fungus ganoderma lucidium. Proteins, 75(2): 524-527. DOI 10.1002/prot.22346. Hughes, J., 1975. Isolation of an endogenous compound from the brain with pharmacological properties similar to morphine. Brain research, 88(2): 295-308. Johansen, J.S., A.K. Harris, D.J. Rychly and A. Ergul, 2005. Oxidative stress and the use of antioxidants in diabetes: Linking basic science to clinical practice. Cardiovascular diabetology, 4(1): 5. DOI 10.1186/1475-2840-4-5. Kaneto, H., Y. Kajimoto, J. Miyagawa, T. Matsuoka, Y. Fujitani, Y. Umayahara, T. Hanafusa, Y. Matsuzawa, Y. Yamasaki and M. Hori, 1999. Beneficial effects of antioxidants in diabetes: Possible protection of pancreatic beta-cells against glucose toxicity. Diabetes, 48(12): 2398-2406. Kino, K., K. Mizumoto, T. Sone, T. Yamaji, J. Watanabe, A. Yamashita, K. Yamaoka, K. Shimizu, K. Ko and H. Tsunoo, 1990. An immunomodulating protein, ling zhi-8 (lz-8) prevents insulitis in non-obese diabetic mice. Diabetologia, 33(12): 713-718. Kino, K., A. Yamashita, K. Yamaoka, J. Watanabe, S. Tanaka, K. Ko, K. Shimizu and H. Tsunoo, 1989. Isolation and characterization of a new immunomodulatory protein, ling zhi-8 (lz-8), from ganoderma lucidium. The Journal of biological chemistry, 264(1): 472-478. Ko, J.L., C.I. Hsu, R.H. Lin, C.L. Kao and J.Y. Lin, 1995. A new fungal immunomodulatory protein, fip-fve isolated from the edible mushroom, flammulina velutipes and its complete amino-acid-sequence. European Journal of Biochemistry, 228(2): 244-249. Available from <Go to ISI>://WOS:A1995QK72200005. DOI 10.1111/j.1432-1033.1995.00244.x. Korhonen, H. and A. Pihlanto, 2006. Bioactive peptides: Production and functionality. International Dairy Journal, 16(9): 945-960. DOI 10.1016/j.idairyj.2005.10.012. Lin, W.H., C.H. Hung, C.I. Hsu and J.Y. Lin, 1997. Dimerization of the n-terminal amphipathic alpha-helix domain of the fungal immunomodulatory protein from ganoderma tsugae (fip-gts) defined by a yeast two-hybrid system and site-directed mutagenesis. Journal of Biological Chemistry, 272(32): 20044-20048. Available from <Go to ISI>://WOS:A1997XQ05900062. DOI 10.1074/jbc.272.32.20044. Lin, Y.L., Y.C. Liang, Y.S. Tseng, H.Y. Huang, S.Y. Chou, R.S. Hseu, C.T. Huang and B.L. Chiang, 2009. An immunomodulatory protein, ling zhi-8, induced activation and maturation of human monocyte-derived dendritic cells by the nf-kappab and mapk pathways. Journal of leukocyte biology, 86(4): 877-889. Available from DOI 10.1189/jlb.0708441. Makrides, S.C., 1996. Strategies for achieving high-level expression of genes in escherichia coli. Microbiological Reviews, 60(3): 512-&. Available from <Go to ISI>://WOS:A1996VF66600004. Maritim, A.C., R.A. Sanders and J.B. Watkins, 3rd, 2003. Diabetes, oxidative stress, and antioxidants: A review. Journal of biochemical and molecular toxicology, 17(1): 24-38. DOI 10.1002/jbt.10058. Martin, K.O., K. Budai and N.B. Javitt, 1993. Cholesterol and 27-hydroxycholesterol 7 alpha-hydroxylation: Evidence for two different enzymes. Journal of lipid research, 34(4): 581-588. Miller, J.F., 1994. [30] bacterial transformation by electroporation. In: Methods in enzymology, P. M. B. Virginia L. Clark, (Ed.). Academic Press: pp: 375-385. Miyasaka, N., H. Inoue, T. Totsuka, R. Koike, K. Kino and H. Tsunoo, 1992. An immunomodulatory protein, ling zhi-8, facilitates cellular interaction through modulation of adhesion molecules. Biochemical and biophysical research communications, 186(1): 385-390. Morikawa, K., I. Kondo, Y. Kanamaru and S. Nagaoka, 2007. A novel regulatory pathway for cholesterol degradation via lactostatin. Biochemical and biophysical research communications, 352(3): 697-702. Available from DOI 10.1016/j.bbrc.2006.11.090. Murasugi, A., S. Tanaka, N. Komiyama, N. Iwata, K. Kino, H. Tsunoo and S. Sakuma, 1991. Molecular cloning of a cdna and a gene encoding an immunomodulatory protein, ling zhi-8, from a fungus, ganoderma lucidum. The Journal of biological chemistry, 266(4): 2486-2493. Nagaoka, S., Y. Futamura, K. Miwa, T. Awano, K. Yamauchi, Y. Kanamaru, K. Tadashi and T. Kuwata, 2001. Identification of novel hypocholesterolemic peptides derived from bovine milk beta-lactoglobulin. Biochemical and biophysical research communications, 281(1): 11-17. Available from DOI 10.1006/bbrc.2001.4298. Paaventhan, P., J.S. Joseph, S.V. Seow, S. Vaday, H. Robinson, K.Y. Chua and P.R. Kolatkar, 2003. A 1.7 angstrom structure of fve, a member of the new fungal immunomodulatory protein family. Journal of Molecular Biology, 332(2): 461-470. Available from <Go to ISI>://WOS:000185306700014. DOI 10.1016/s0022-2836(03)00923-9. Pan, D., H. Guo, B. Zhao and J. Cao, 2011. The molecular mechanisms of interactions between bioactive peptides and angiotensin-converting enzyme. Bioorganic & medicinal chemistry letters, 21(13): 3898-3904. Available from DOI 10.1016/j.bmcl.2011.05.033. Qian, Z.J., W.K. Jung and S.K. Kim, 2008. Free radical scavenging activity of a novel antioxidative peptide purified from hydrolysate of bullfrog skin, rana catesbeiana shaw. Bioresource technology, 99(6): 1690-1698. DOI 10.1016/j.biortech.2007.04.005. Rajapakse, N., E. Mendis, W.K. Jung, J.Y. Je and S.K. Kim, 2005. Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties. Food Research International, 38(2): 175-182. Available from <Go to ISI>://WOS:000226447800009. DOI 10.1016/j.foodres.2004.10.002. Rengarajan, J., S.J. Szabo and L.H. Glimcher, 2000. Transcriptional regulation of th1/th2 polarization. Immunology today, 21(10): 479-483. Rice-Evans, C.A. and A.T. Diplock, 1993. Current status of antioxidant therapy. Free radical biology & medicine, 15(1): 77-96. Rodriguez, J.M., M.I. Martinez, N. Horn and H.M. Dodd, 2003. Heterologous production of bacteriocins by lactic acid bacteria. International Journal of Food Microbiology, 80(2): 101-116. Available from <Go to ISI>://WOS:000179031900001. DOI 10.1016/s0168-1605(02)00153-8. Saito, K., D.H. Jin, T. Ogawa, K. Muramoto, E. Hatakeyama, T. Yasuhara and K. Nokihara, 2003. Antioxidative properties of tripeptide libraries prepared by the combinatorial chemistry. Journal of Agricultural and Food Chemistry, 51(12): 3668-3674. Available from <Go to ISI>://WOS:000183224500029. DOI 10.1021/jf021191n. Sarmadi, B.H. and A. Ismail, 2010. Antioxidative peptides from food proteins: A review. Peptides, 31(10): 1949-1956. Available from DOI 10.1016/j.peptides.2010.06.020. Schallmey, M., A. Singh and O.P. Ward, 2004. Developments in the use of bacillus species for industrial production. Canadian journal of microbiology, 50(1): 1-17. Available from DOI 10.1139/w03-076. Schellenberger, V., H.D. Jakubke, N.P. Zapevalova and Y.V. Mitin, 1991. Protease-catalyzed peptide synthesis using inverse substrates: The influence of reaction conditions on the trypsin acyl transfer efficiency. Biotechnology and bioengineering, 38(1): 104-108. DOI 10.1002/bit.260380114. Setlow, P., 2006. Spores of bacillus subtilis: Their resistance to and killing by radiation, heat and chemicals. Journal of applied microbiology, 101(3): 514-525. Available from DOI 10.1111/j.1365-2672.2005.02736.x. Tanaka, S., K. Ko, K. Kino, K. Tsuchiya, A. Yamashita, A. Murasugi, S. Sakuma and H. Tsunoo, 1989. Complete amino acid sequence of an immunomodulatory protein, ling zhi-8 (lz-8). An immunomodulator from a fungus, ganoderma lucidium, having similarity to immunoglobulin variable regions. The Journal of biological chemistry, 264(28): 16372-16377. Tjalsma, H., A. Bolhuis, J.D.H. Jongbloed, S. Bron and J.M. van Dijl, 2000. Signal peptide-dependent protein transport in bacillus subtilis: A genome-based survey of the secretome. Microbiology and Molecular Biology Reviews, 64(3): 515-+. Available from <Go to ISI>://WOS:000089249600004. DOI 10.1128/mmbr.64.3.515-547.2000. Tomaszewski, M., K.M. Stepien, J. Tomaszewska and S.J. Czuczwar, 2011. Statin-induced myopathies. Pharmacological Reports, 63(4): 859-866. Available from <Go to ISI>://WOS:000297530600002. Wang, J.P., C.M. Yeh and Y.C. Tsai, 2006. Improved subtilisin yab production in bacillus subtilis using engineered synthetic expression control sequences. J Agric Food Chem, 54(25): 9405-9410. DOI 10.1021/jf061982f. Westers, L., H. Westers and W.J. Quax, 2004. Bacillus subtilis as cell factory for pharmaceutical proteins: A biotechnological approach to optimize the host organism. Biochimica Et Biophysica Acta-Molecular Cell Research, 1694(1-3): 299-310. Available from <Go to ISI>://WOS:000226213500022. DOI 10.1016/j.bbammcr.2004.02.011. Wu, S.C., J.C. Yeung, Y.J. Duan, R.Q. Ye, S.J. Szarka, H.R. Habibi and S.L. Wong, 2002. Functional production and characterization of a fibrin-specific single-chain antibody fragment from bacillus subtilis: Effects of molecular chaperones and a wall-bound protease on antibody fragment production. Applied and Environmental Microbiology, 68(7): 3261-3269. Available from <Go to ISI>://WOS:000176631600010. DOI 10.1128/aem.68.7.3261-3269.2002. Yeh, C.M., C.K. Yeh, X.Y. Hsu, Q.M. Luo and M.Y. Lin, 2008. Extracellular expression of a functional recombinant ganoderma lucidium immunomodulatory protein by bacillus subtilis and lactococcus lactis. Appl Environ Microbiol, 74(4): 1039-1049. DOI 10.1128/aem.01547-07.
枯草桿菌為一革蘭氏陽性、兼性厭氧之產孢桿狀細菌。在遺傳工程上經常被用來作為表現異源蛋白之宿主,其原因如下:(1) 培養容易,(2) 被認可為 GRAS 級菌種,(3) 具有 DNA 轉型能力,(4) 具備強力之蛋白分泌能力。
生物活性胜肽指的是來自於動物或植物之蛋白質,經由蛋白酶水解後所釋出之短鍊胜肽,其具有調節人體生理機能之活性。自大豆中分離出來之抗氧化胜肽:Leu-Leu- Pro-His-His (LLPHH),經研究發現具有抑制脂質過氧化之能力,且其抗氧化活性與胜肽 C 端之 -His-His 有著密切之關聯。Ile-Ile-Ala-Glu-Lys (IIAEK),名為 Lactostatin,為一自牛乳 β-乳球蛋白中所分離出來之胜肽。此胜肽具有降低膽固醇之活性,其作用機制為活化肝臟細胞中特異性存在之酵素:cholesterol 7α-hydroxylase 基因之表現,該酵素負責膽固醇與膽酸轉換過程之速率決定步驟。
靈芝免疫調節蛋白(Ling-Zhi 8;LZ-8)為一自靈芝(Ganoderma lucidum)菌絲體中分離出來之蛋白。其生理活性包括有免疫調節、抗腫瘤、減緩器官移植之排斥作用等。研究指出其免疫調節活性與其雙體結構有密切之關聯性。本實驗室先前已成功於枯草桿菌表現重組靈芝免疫調節蛋白(recombinant LZ-8;rLZ-8),並且證實重組靈芝免疫調節蛋白具有刺激多種細胞激素分泌之活性。之後本實驗室進一步嘗試於rLZ-8上融合抗氧化胜肽LLPHH或降膽固醇胜肽IIAEK,以期能獲得多重生理活性之蛋白,而在設計上也於胜肽與rLZ-8間導入蛋白酶切位以使胜肽能透過蛋白酶之水解而自融合蛋白上釋出。先前之設計為融合三重複之LLPHH於 rLZ-8上以及三重複之LLPHH與三重複之IIAEK於 rLZ-8上,並且導入連接子、必需胺基酸及蛋白酶切位胺基酸,但此兩蛋白卻不易在枯草桿菌表現及純化,推論其原因可能為所接上之胺基酸序列影響到rLZ-8 之構型,使融合蛋白不易被表現或被枯草桿菌胞內之蛋白酶降解系統所分解。
本實驗嘗試改進融合蛋白所接上之胺基酸序列。LLPHH及IIAEK均融合一重複或二重複於 rLZ-8上,並導入trypsin或pepsin切位胺基酸,將四種融合蛋白命名為rLZHO、 rLZHO2、rLZHC 及rLZHC2。結果顯示此四個融合蛋白均能於枯草桿菌表現並被純化到,分別為130μg/ml、30μg/ml、270μg/ml、220μg/ml。在經由蛋白酶作用後生物活性胜肽能自融合蛋白上釋出,之後將兩種生物活性胜肽以高效能液相層析分析後發現兩者之身分均與化學合成之標準品相同。抗氧化胜肽之活性測試結果顯示來自融合蛋白 rLZHO 之 LLPHH 可有效抑制亞麻油酸之自氧化反應,且於 10-4 M 下其抑制效果較 BHA 佳。


Bacillus subtilis is a Gram-positive, facultative anaerobic, endospore forming rod-shaped bacterium. It has been widely used in production of recombinant protein due to it’s characteristics such as easy to culture, as a GRAS bacterium, has DNA transforming ability and powerful protein secretion capacity.
Bioactive peptides are designated as peptides released from animal or plant proteins after enzymatic digestion, with regulatory functions in human body. Leu-Leu- Pro-His-His (LLPHH), isolated from soybean protein, is an antioxidative peptide which can inhibit lipid peroxidation. Its activity is highly related to the C-terminal –His-His. Ile-Ile-Ala-Glu-Lys (IIAEK), termed as lactostatin, is a hypocholesterolemic peptide isolated from bovine milk β-lactoglobulin. Its hypocholesterolemic mechanism is to activate the gene expression of a liver specific enzyme, cholesterol 7α-hydroxylase, which is the rate-limiting enzyme in the pathway of cholesterol and bile acid conversion.
Ling-Zhi 8 (LZ-8) is a protein isolated from the mycelium of Ganoderma lucidum. Its biological activities include immunomodulatory responses, antitumor activity, alleviation of transplant rejection. Research showed that the activity is highly related to its dimer form. In our previous study, recombinant LZ-8 was successfully expressed by our Bacillus subtilis system, and the cytokines’ secretion stimulation and immunomodulatory activity were proved. After that we tried to fuse rLZ-8 with LLPHH or IIAEK, and also introduced protease cleavage sites between bioactive peptides and rLZ-8 so that the bioactive peptides can be released from the protein by protease digestion. Previously we fused rLZ-8 with three copies of LLPHH in one designation and three copies of LLPHH together with three copies of IIAEK in another, and also added linker, essential amino acids and protease cleavage sites in the fusion sequence. But the expressions were not so successful by the B. subtilis system. It is possible that the amino acid sequence fused with rLZ-8 may interfere the conformation of rLZ-8, so the fusion proteins were hard to express in B. subtilis or degraded by its intracellular protease system.
In this study, we tried to change the amino acid sequence fused to rLZ-8. We fused one copy or two copies of LLPHH to rLZ-8 with trypsin or pepsin cleavage sites, and these two fusion proteins were designated as rLZHO and rLZHO2. In IIAEK we constructed same copies and designated as rLZHC and rLZHC2. These four fusion proeins were successfully expressed in B. subtilis WB800 and could be purified to obtain 130μg/ml, 30μg/ml, 270μg/ml, 220μg/ml proteins, respectively. After protease treatment the peptides could be released from the fusion proteins. The HPLC analysis proved that the bioactive peptides were the same as standards, and the rLZ-8 was in a dimer form that indicated the bioactivity of rLZ-8. The result of antioxidative activity assay showed that the LLPHH from rLZHO can inhibit the autoxidation of linoleic acid, and the antioxidative activity is higer than BHA in 10-4 M.

Keywords: Bacillus subtilis, Ling-Zhi 8, dimer form, antioxidative peptide, hypocholesterolemic peptide, fusion protein
其他識別: U0005-2008201218055300
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