应用定向进化获取受体高亲和性hEGF突变株E40V及其特性的评价
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摘要
人表皮生长因子(human epidermal growth factor,hEGF)是一种由53个氨基酸构成的单体多肽。该蛋白作为有效的促有丝分裂因子,通过和细胞表面受体EGFR特异性结合来影响细胞的生长,增殖和分化。hEGF基因的失调与某些癌症的生长和发育有关,其蛋白本身对于表皮损伤和溃疡具有良好的修复功能。但是由于其半衰期较短且会造成受体表达下调,因此有必要对其进行改良。通过StEP法对人源和猪源EGF因子进行定向化改造,通过构建噬菌体展示文库和ELISA筛选方式获得一株对于受体具有较高亲和力的突变株E40V,并使用MTT、圆二色谱和细胞划痕对该突变株的促细胞增殖,迁移能力以及温度,pH耐受性进行了探究。结果显示,E40V相较于野生型EGF,其受体亲和性显著提高,对于正常细胞的促增殖能力也有所强化,而对于温度,pH耐受情况则与野生型相差不大。本研究不仅为短肽的定向进化提供了一个范例,也为EGF的实际应用打下基础。
Human epidermal growth factor(hEGF) is a monomeric polypeptide consisting of 53 amino acids. The protein acts as an active mitogenic factor and affects cell growth, proliferation and differentiation by specifically binding to its cell surface receptor EGFR. The dysregulation of the hEGF gene is associated with the growth and development of certain cancers. Although hEGF protein shows a quick repair function for epidermal tissue damage and ulceration disease, some ambiguities such as short half-life and EGFR down-regulation still remain. In this study, the human EGF and porcine EGF factor were modified via directed evlotion in vitro by the staggered extension process(StEP), in which a mutant E40V with high affinity against EGFR receptor was obtained using phage display library construction and ELISA screening. Then we used MTT, circular dichroism(CD) and wound healing assay to analyze the A431 and NIH3T3 cell proliferation, migration ability, temperature and pH tolerance of the mutant protein respectively. We found that the affinity of E40V for EGFR was significantly higher than that of wild-type EGF and the mutant could also promote the proliferation of normal cells NIH3T3, while the temperature tolerance and pH tolerance were similar to those of wild-type. Our DNA shuffling EGF with targeting properties and long half-life may provide a new application foreground for regulations of the normal cell growth and cancer targeting therapy.
Human epidermal growth factor(hEGF) is a monomeric polypeptide consisting of 53 amino acids. The protein acts as an active mitogenic factor and affects cell growth, proliferation and differentiation by specifically binding to its cell surface receptor EGFR. The dysregulation of the hEGF gene is associated with the growth and development of certain cancers. Although hEGF protein shows a quick repair function for epidermal tissue damage and ulceration disease, some ambiguities such as short half-life and EGFR down-regulation still remain. In this study, the human EGF and porcine EGF factor were modified via directed evlotion in vitro by the staggered extension process(StEP), in which a mutant E40V with high affinity against EGFR receptor was obtained using phage display library construction and ELISA screening. Then we used MTT, circular dichroism(CD) and wound healing assay to analyze the A431 and NIH3T3 cell proliferation, migration ability, temperature and pH tolerance of the mutant protein respectively. We found that the affinity of E40V for EGFR was significantly higher than that of wild-type EGF and the mutant could also promote the proliferation of normal cells NIH3T3, while the temperature tolerance and pH tolerance were similar to those of wild-type. Our DNA shuffling EGF with targeting properties and long half-life may provide a new application foreground for regulations of the normal cell growth and cancer targeting therapy.
引文
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[23] ABDOLALIZADEH J,NOURI M,ZOLBANIN J M,et al.Targeting cytokines:production and characterization of anti-TNF-alpha scFvs by phage display technology [J].Curr Pharm Des,2013,19(15):2839-2847.
[24] ZHAO Y,WANG Q,JIN Y,et al.Discovery and Characterization of a high-affinity small peptide ligand,H1,targeting FGFR2IIIc for skin wound healing [J].Cell Physiol Biochem,2018,49(3):1033-1048.
[25] MATTIASSON B,KUMAR A,IVANOV A E,et al.Metal-chelate affinity precipitation of proteins using responsive polymers [J].Nat Protoc,2007,2(1):213-220.
[26] SMITH P K,KROHN R I,HERMANSON G T,et al.Measurement of protein using bicinchoninic acid [J].Anal Biochem,1985,150(1):76-85.
[27] HIKITA S,YOTSUMOTO F,FUMAKI T,et al.Assessment of HB-EGF Levels in Peritoneal Fluid and Serum of Ovarian Cancer Patients using ELISA [J].Anticancer Research,2011,31(7):2553-2559.
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[30] KANG J,TADA S,SAKURAGI M,et al.An epidermal growth factor derivative with binding affinity for hydroxyapatite and titanium surfaces [J].Biomaterials,2013,34(38):9747-9753.
[31] MILES A J,WALLACE B A.CDtoolX,a downloadable software package for processing and analyses of circular dichroism spectroscopic data,protein science [CP/OL].(2018-05-04) [2019-1-23].http://www.cdtools.cryst.bbk.ac.uk.DOI:10.1002/pro.3474.
[32] COCO W M,LEVINSON W E,CRIST M J,et al.DNA shuffling method for generating highly recombined genes and evolved enzymes [J].Nat Biotechnol,2001,19(4):354-359.
[33] SHE X,HORVATH J E,JIANG Z,et al.The structure and evolution of centromeric transition regions within the human genome [J].Nature,2004,430(7002):857-864.
[34] ZHANG H,KONG X,ZHANG J.New strategies of protein engineering-directed evolution of enzymein vitro [J].Chinese Science Bulletin,1999,44(18):1641-1648.
[35] YU C,HALE J,RITCHIE K,et al.Receptor overexpression or inhibition alters cell surface dynamics of EGF-EGFR interaction:new insights from real-time single molecule analysis [J].Biochem Biophys Res Commun,2009,378(3):376-82.
[36] HUANG P,XU X,WANG L,et al.The role of EGF-EGFR signalling pathway in hepatocellular carcinoma inflammatory microenvironment [J].J Cell Mol Med,2014,18(2):218-230.
[37] VANHERCKE T,AMPE C,TIRRY L,et al.Reducing mutational bias in random protein libraries [J].Anal Biochem,2005,339(1):9-14.
[38] MULLENBACH G T,CHIU C Y,GYENES A,et al.Modification of a receptor-binding surface of epidermal growth factor (EGF):analogs with enhanced receptor affinity at low pH or at neutrality [J].Protein Eng,1998,11(6):473-480.
[39] HE M,YANG Z Y,NIE Y F,et al.A new type of class I bacterial 5-enopyruvylshikimate-3-phosphate synthase mutants with enhanced tolerance to glyphosate [J].Biochim Biophys Acta,2001,1568(1):1-6.
[40] WANG Y,XU Y,MA X,et al.Extraction,purification,characterization and antioxidant activities of polysaccharides from Zizyphus jujuba cv.Linzexiaozao [J].Int J Biol Macromol,2018,118(Pt B):2138-2148.
[41] HOGREFE H H,CLINE J,YOUNGBLOOD G L,et al.Creating randomized amino acid libraries with the quik change multi site-directed mutagenesis kit [J].Biotechniques,2002,33(5):1158-1160.
[42] GOENAGA A L,ZHOU Y,LEGAY C,et al.Identification and characterization of tumor antigens by using antibody phage display and intrabody strategies [J].Mol Immunol,2007,44(15):3777-3788.
[43] HOOGENBOOM H R.Selecting and screening recombinant antibody libraries [J].Nat Biotechnol,2005,23(9):1105-1116.
[44] LIANG S,LIN T,DING J,et al.Screening and identification of vascular-endothelial-cell-specific binding peptide in gastric cancer [J].J Mol Med (Berl),2006,84(9):764-773.
[45] KRAG D N,SHUKLA G S,SHEN G P,et al.Selection of tumor-binding ligands in cancer patients with phage display libraries [J].Cancer Res,2006,66(15):7724-7733.
[46] CHOUDHRY V,ZHANG M Y,SIDOROV I A,et al.Cross-reactive HIV-1 neutralizing monoclonal antibodies selected by screening of an immune human phage library against an envelope glycoprotein (gp140) isolated from a patient (R2) with broadly HIV-1 neutralizing antibodies [J].Virology,2007,363(1):79-90.
[47] BARRY M A,DOWER W J,JOHNSTON S A.Toward cell-targeting gene therapy vectors:Selection of cell-binding peptides from random peptide-presenting phage libraries [J].Nature Medicine,1996,2(3):299-305.
[48] WEBB S E,ROBERTS S K,NEEDHAM S R,et al.Single-molecule imaging and fluorescence lifetime imaging microscopy show different structures for high-and low-affinity epidermal growth factor receptors in A431 cells [J].Biophys J,2008,94(3):803-819.
[49] MADAN E,PRASAD S,ROY P,et al.Regulation of apoptosis by resveratrol through JAK/STAT and mitochondria mediated pathway in human epidermoid carcinoma A431 cells [J].Biochem Biophys Res Commun,2008,377(4):1232-1237.
[50] STUART M J,NAGEL R L.Sickle-cell disease [J].Lancet,2004,364(9442):1343-1360.
[2] CURY P R,de ARAUJO V C,CANAVEZ F,et al.The effect of epidermal growth factor on matrix metalloproteinases and tissue inhibitors of metalloproteinase gene expression in cultured human gingival fibroblasts [J].Arch Oral Biol,2007,52(6):585-590.
[3] CALDWELL M A,HE X,WILKIE N,et al.Growth factors regulate the survival and fate of cells derived from human neurospheres [J].Nat Biotechnol,2001,19(5):475-479.
[4] OGISO H,ISHITANI R,NUREKI O,et al.Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains [J].Cell,2002,110(6):775-787.
[5] HERMANN P M,van KESTEREN R E,WILDERING W C,et al.Neurotrophic actions of a novel molluscan epidermal growth factor [J].J Neurosci,2000,20(17):6355-6364.
[6] TEDESCO K L,LOCKHART A C,BERLIN J D.The epidermal growth factor receptor as a target for gastrointestinal cancer therapy [J].Current Treatment Options in Oncology,2004,5(5):393-403.
[7] LYNCH T J,BELL D W,SORDELLA R,et al.Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib [J].N Engl J Med,2004,350(21):2129-2139.
[8] REDDY C C,NIYOGI S K,WELLS A,et al.Engineering epidermal growth factor for enhanced mitogenic potency [J].Nat Biotechnol,1996,14(13):1696-1699.
[9] SUTTON R,WARD W G,RAPHAEL K A,et al.Growth factor expression in skin during wool follicle development [J].Comp Biochem Physiol B Biochem Mol Biol,1995,110(4):697-705.
[10] CALNAN D P,FAGBEMI A,BERLANGA-ACOSTA J,et al.Potency and stability of C terminal truncated human epidermal growth factor [J].Gut,2000,47(5):622-627.
[11] PLAYFORD R J,MARCHBANK T,CALNAN D P,et al.Epidermal growth factor is digested to smaller,less active forms in acidic gastric juice [J].Gastroenterology,1995,108(1):92-101.
[12] SENDEROFF R I,WOOTTON S C,BOCTOR A M,et al.Aqueous stability of human epidermal growth factor 1-48 [J].Pharm Res,1994,11(12):1712-1720.
[13] AGUIRRE A,DUPREE J L,MANGIN J M,et al.A functional role for EGFR signaling in myelination and remyelination [J].Nat Neurosci,2007,10(8):990-1002.
[14] SEBASTIAN S,SETTLEMAN J,RESHKIN S J,et al.The complexity of targeting EGFR signalling in cancer:from expression to turnover [J].Biochim Biophys Acta,2006,1766(1):120-139.
[15] FOX R J,HUISMAN G W.Enzyme optimization:moving from blind evolution to statistical exploration of sequence-function space [J].Trends Biotechnol,2008,26(3):132-138.
[16] STEMMER W P.DNA shuffling by random fragmentation and reassembly:in vitro recombination for molecular evolution [J].Proc Natl Acad Sci U S A,1994,91(22):10747-10751.
[17] CURRIN A,SWAINSTON N,DAY P J,et al.Synthetic biology for the directed evolution of protein biocatalysts:navigating sequence space intelligently [J].Chem Soc Rev,2015,44(5):1172-1239.
[18] COCO W M,ENCELL L P,LEVINSON W E,et al.Growth factor engineering by degenerate homoduplex gene family recombination [J].Nat Biotechnol,2002,20(12):1246-1250.
[19] ZHAO H,GIVER L,SHAO Z,et al.Molecular evolution by staggered extension process (StEP) in vitro recombination [J].Nat Biotechnol,1998,16(3):258-261.
[20] NELSON R S,VALADON P.A universal phage display system for the seamless construction of Fab libraries [J].J Immunol Methods,2017,(450):41-49.
[21] ZHAO H,ZHA W.In vitro 'sexual' evolution through the PCR-based staggered extension process (StEP) [J].Nat Protoc,2006,1(4):1865-1871.
[22] WANG X,ZHANG W,TANG J,et al.LINC01225 promotes occurrence and metastasis of hepatocellular carcinoma in an epidermal growth factor receptor-dependent pathway [J].Cell Death Dis,2016,7:e2130.
[23] ABDOLALIZADEH J,NOURI M,ZOLBANIN J M,et al.Targeting cytokines:production and characterization of anti-TNF-alpha scFvs by phage display technology [J].Curr Pharm Des,2013,19(15):2839-2847.
[24] ZHAO Y,WANG Q,JIN Y,et al.Discovery and Characterization of a high-affinity small peptide ligand,H1,targeting FGFR2IIIc for skin wound healing [J].Cell Physiol Biochem,2018,49(3):1033-1048.
[25] MATTIASSON B,KUMAR A,IVANOV A E,et al.Metal-chelate affinity precipitation of proteins using responsive polymers [J].Nat Protoc,2007,2(1):213-220.
[26] SMITH P K,KROHN R I,HERMANSON G T,et al.Measurement of protein using bicinchoninic acid [J].Anal Biochem,1985,150(1):76-85.
[27] HIKITA S,YOTSUMOTO F,FUMAKI T,et al.Assessment of HB-EGF Levels in Peritoneal Fluid and Serum of Ovarian Cancer Patients using ELISA [J].Anticancer Research,2011,31(7):2553-2559.
[28] GraphPad Software.GraphPad prism version 8.0.0 for Windows [CP/OL].San Diego,California USA:GraphPad Software,Inc.(2018-04-14) [2019-1-3].https://www.graphpad.com/.
[29] HARDWICKE J,MOSELEY R,STEPHENS P,et al.Bioresponsive dextrin-rhEGF conjugates:in vitro evaluation in models relevant to its proposed use as a treatment for chronic wounds [J].Mol Pharm,2010,7(3):699-707.
[30] KANG J,TADA S,SAKURAGI M,et al.An epidermal growth factor derivative with binding affinity for hydroxyapatite and titanium surfaces [J].Biomaterials,2013,34(38):9747-9753.
[31] MILES A J,WALLACE B A.CDtoolX,a downloadable software package for processing and analyses of circular dichroism spectroscopic data,protein science [CP/OL].(2018-05-04) [2019-1-23].http://www.cdtools.cryst.bbk.ac.uk.DOI:10.1002/pro.3474.
[32] COCO W M,LEVINSON W E,CRIST M J,et al.DNA shuffling method for generating highly recombined genes and evolved enzymes [J].Nat Biotechnol,2001,19(4):354-359.
[33] SHE X,HORVATH J E,JIANG Z,et al.The structure and evolution of centromeric transition regions within the human genome [J].Nature,2004,430(7002):857-864.
[34] ZHANG H,KONG X,ZHANG J.New strategies of protein engineering-directed evolution of enzymein vitro [J].Chinese Science Bulletin,1999,44(18):1641-1648.
[35] YU C,HALE J,RITCHIE K,et al.Receptor overexpression or inhibition alters cell surface dynamics of EGF-EGFR interaction:new insights from real-time single molecule analysis [J].Biochem Biophys Res Commun,2009,378(3):376-82.
[36] HUANG P,XU X,WANG L,et al.The role of EGF-EGFR signalling pathway in hepatocellular carcinoma inflammatory microenvironment [J].J Cell Mol Med,2014,18(2):218-230.
[37] VANHERCKE T,AMPE C,TIRRY L,et al.Reducing mutational bias in random protein libraries [J].Anal Biochem,2005,339(1):9-14.
[38] MULLENBACH G T,CHIU C Y,GYENES A,et al.Modification of a receptor-binding surface of epidermal growth factor (EGF):analogs with enhanced receptor affinity at low pH or at neutrality [J].Protein Eng,1998,11(6):473-480.
[39] HE M,YANG Z Y,NIE Y F,et al.A new type of class I bacterial 5-enopyruvylshikimate-3-phosphate synthase mutants with enhanced tolerance to glyphosate [J].Biochim Biophys Acta,2001,1568(1):1-6.
[40] WANG Y,XU Y,MA X,et al.Extraction,purification,characterization and antioxidant activities of polysaccharides from Zizyphus jujuba cv.Linzexiaozao [J].Int J Biol Macromol,2018,118(Pt B):2138-2148.
[41] HOGREFE H H,CLINE J,YOUNGBLOOD G L,et al.Creating randomized amino acid libraries with the quik change multi site-directed mutagenesis kit [J].Biotechniques,2002,33(5):1158-1160.
[42] GOENAGA A L,ZHOU Y,LEGAY C,et al.Identification and characterization of tumor antigens by using antibody phage display and intrabody strategies [J].Mol Immunol,2007,44(15):3777-3788.
[43] HOOGENBOOM H R.Selecting and screening recombinant antibody libraries [J].Nat Biotechnol,2005,23(9):1105-1116.
[44] LIANG S,LIN T,DING J,et al.Screening and identification of vascular-endothelial-cell-specific binding peptide in gastric cancer [J].J Mol Med (Berl),2006,84(9):764-773.
[45] KRAG D N,SHUKLA G S,SHEN G P,et al.Selection of tumor-binding ligands in cancer patients with phage display libraries [J].Cancer Res,2006,66(15):7724-7733.
[46] CHOUDHRY V,ZHANG M Y,SIDOROV I A,et al.Cross-reactive HIV-1 neutralizing monoclonal antibodies selected by screening of an immune human phage library against an envelope glycoprotein (gp140) isolated from a patient (R2) with broadly HIV-1 neutralizing antibodies [J].Virology,2007,363(1):79-90.
[47] BARRY M A,DOWER W J,JOHNSTON S A.Toward cell-targeting gene therapy vectors:Selection of cell-binding peptides from random peptide-presenting phage libraries [J].Nature Medicine,1996,2(3):299-305.
[48] WEBB S E,ROBERTS S K,NEEDHAM S R,et al.Single-molecule imaging and fluorescence lifetime imaging microscopy show different structures for high-and low-affinity epidermal growth factor receptors in A431 cells [J].Biophys J,2008,94(3):803-819.
[49] MADAN E,PRASAD S,ROY P,et al.Regulation of apoptosis by resveratrol through JAK/STAT and mitochondria mediated pathway in human epidermoid carcinoma A431 cells [J].Biochem Biophys Res Commun,2008,377(4):1232-1237.
[50] STUART M J,NAGEL R L.Sickle-cell disease [J].Lancet,2004,364(9442):1343-1360.