成功大學博碩士論文系統

簡易檢索 / 詳目顯示

回結果列表

研究生：	游凱丞 Yu, Kai-Cheng
論文名稱：	優化第三型纖維黏蛋白的第十個模組片段之EGFR專一性拮抗劑 Optimization of EGFR-specific Antagonist Using the Tenth Module of Fibronectin Type III Domain
指導教授：	莊偉哲 Chuang, Woei-Jer
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學暨分子生物學研究所 Department of Biochemistry and Molecular Biology
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	96
中文關鍵詞：	表皮生長因子受體、表皮生長因子受體抑制劑、纖維黏蛋白、Adnectin 1 、蛋白質工程、DbD2 、溶解度、熱穩定性
外文關鍵詞：	EGFR, EGFR inhibitor, Fibronectin, Adnectin 1, Protein engineering, DbD2, Solubility, Thermostability
相關次數：	點閱：86 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

表皮生長因子受體（EGFR）屬於受體酪氨酸激酶家族，具有調節細胞增殖、遷移、侵襲、血管生成和抑制細胞凋亡等等重要的功能。在許多癌症細胞中都有發現EGFR過量表現的現象，其中包括肺癌、乳癌以及大腸癌等常見癌症，因此在過去研究中，EGFR一直以來都是一個很重要的目標，並且目前市面上也有許多藥物是針對EGFR進行設計，但是這些藥物卻有副作用，甚至產生抗藥性的現象，因此，針對EGFR進行辨識的藥物開發還是一個很重要的議題。E1和E4是衍生自人類第三型纖維黏蛋白的第十個模組（10Fn3）的EGFR拮抗劑，能夠抑制EGFR的磷酸化進而阻斷下游信號的傳遞，然而，卻存在著穩定性的問題。為了提高它們的熱穩定性、溶解度和活性，我們將E1和E4辨識EGFR關鍵的三個loop（BC，DE和FG）序列導入到10Fn3中，並使用生物資訊工具：Disulfide by design 2.0（Dbd2）設計雙硫鍵引入其中。在我們實驗室先前的研究當中，我們發現χ3角度這個因子對於形成雙硫鍵當中扮演很重要的角色，另外，也發現loop和βstrand之間的雙硫鍵鍵結可以有效幫助蛋白結構更穩定，進而表現出最高的熱穩定性。因此根據上述發現，我將E1以及E4分別接上L9C-S95C、G38C-V46C和E39C-G42C雙硫鍵進行測試。此外，我也在E4和E4 G38C-V46的C端加上Fn在自然界中的linker進行測試。在差示掃描量熱法（DSC）分析結果中，E1 L9C/S95C、G38C/V46C和E39C/G42C的熔解溫度（Tm）值分別為70.7℃，84.7℃，87.2℃和78.6℃。而E4 L9C/S95C、G38C/V46C和E39C/G42C的Tm值分別為71.4/84.1℃，90.0℃，88.3℃和56.9/79.2℃。E4-tail和E4 G38C/V46C-tail的Tm值分別為76.6℃和87.3℃。從這些結果中我們發現，接上L9C-S95C或G38C-V46C雙硫鍵可以提高E1和E4的熱穩定性，Tm值提升14-17℃。在溶解度方面，E4 L9C/S95C和G38C/V46在PBS的溶解度為145.2和36.5mg/ml。E1 L9C/S95C和G38C/V46C的溶解度為79.4和54.0 mg/ml。而E4-tail和E4 G38C/V46C-tail的溶解度分別為207.9 mg/ml和201.6mg/ml。結果顯示，不論是接上L9C-S95C或G38C-V46C雙硫鍵，還是在蛋白質C端加入linker都可以增加E1和E4的溶解度。而在使用A549肺腺癌肺泡基底上皮細胞，測試抑制EGF所誘導的細胞遷移研究中，E4 G38C/V46C表現出比E4高至少10倍的抑制能力。在測試蛋白質與EGFR的結合親和力方面，則是使用A549細胞進行流式細胞儀分析。結果同樣發現，不僅接上L9C-S95C或G38C-V46C雙硫鍵，在蛋白質C端加入linker也可以增加E1和E4對EGFR的結合親和力。本篇研究的結果將作為設計辨識EGFR和同時辨識整合蛋白以及EGFR的10Fn3突變體的基礎。

The epidermal growth factor receptor (EGFR) belongs to the family of tyrosine kinase receptors and regulates cell proliferation, migration, invasion, angiogenesis, and inhibition of cell apoptosis. Overexpression of EGFR is found in many cancer cell types, including lung cancer, glioblastoma, as well as head and neck squamous cell carcinoma. E1 and E4 are EGFR antagonists derived from the tenth type III domain of human fibronectin (10Fn3) and inhibit EGFR phosphorylation and downstream signaling cascade. To improve their thermostability, solubility, and activity, we incorporated three binding loops (BC, DE, and FG) of E1 and E4 into wild-type 10Fn3 and introduced disulfide bond into them using the bioinformatics tool, Disulfide by design 2.0 (Dbd2). Our previous study showed that the introduction of disulfide bond using the χ3 torsion angles criteria gave the best result, and the disulfide bond between loop and β strand exhibited the highest thermostability. I therefore expressed E1, E4, and their mutants by introducing L9C-S95C, G38C-V46C, and E39C-G42C disulfide bonds. Besides, I construct 7 amino acids into E4 and E4-G38C-V46 C-terminal. The analyses using differential scanning calorimetry (DSC) showed that the melting temperature (Tm) values of E1 L9C/S95C, G38C/V46C, and E39C/G42C mutants were 70.7℃, 84.7℃, 87.2℃, and 78.6℃. The Tm values of E4 L9C/S95C, G38C/V46C, and E39C/G42C mutants were 71.4/84.1℃, 90.0℃, 88.3℃, and 56.9/79.2℃. The Tm values of E4-tail and E4 G38C/V46C-tail were 76.6℃ and 87.3℃.These results showed that the incorporation of either L9C-S95C or G38C-V46C disulfide bond can increase the thermostability of E1 and E4 with the Tm values up to 14-17℃. The solubility of E4 L9C/S95C and G38C/V46C mutants were 145.2, 36.5 mg/ml. The solubility of E1 L9C/S95C and G38C/V46C mutants were 79.4, 54.0 mg/ml. The solubility of E4-tail and E4 G38C/V46C-tail was 207.9 mg/ml and 201.6 mg/ml, respectively. The analysis showed that not only the incorporation of either L9C-S95C or G38C-V46C disulfide bond, but C-terminal tail can increase the solubility of E1 and E4. The inhibition of EGF-induced cell migration using A549 lung adenocarcinomic human alveolar basal epithelial cells showed that E4 G38C/V46C mutant exhibited at least 10-fold higher inhibitory activity than E4. The bindings of E4 10Fn3 mutants to EGFR were examined using the flow cytometry analysis of A549 cell. The analysis showed that not only the incorporation of either L9C-S95C or G38C-V46C disulfide bond, but C-terminal tail can increase the bindings of E1 and E4 to EGFR. The results of this study will serve as the basis for the design of anti-EGFR and bispecific anti-integrin and EGFR 10Fn3 mutants.

CHINESE ABSTRACT..............................................................................I
ABSTRACT......................................................................................III
ACKNOWLEDGMENT................................................................................V
TABLE OF CONTENTS.............................................................................VII
LIST OF TABLES................................................................................X
LIST OF FIGURES...............................................................................XI
ABBREVIATIONS.................................................................................XII
CHAPTER 1 INTRODUCTION........................................................................1
1	Rationale.............................................................................1
2	Lung Cancer...........................................................................2
2.1	Overview of Lung Cancer...............................................................2
2.2	Epidermal Growth Factor Receptor (EGFR) as Therapeutic Target........................................................................................2
2.3	Blockage of Epidermal Growth Factor Receptor (EGFR)	..............................................................................................3
3	Fibronectin as the Protein Scaffold for Drug Design...................................4
3.1	Fibronectin (Fn)......................................................................4
3.2	Fibronectin Type III Domain (Fn3).....................................................5
3.3	10Fn3 as the Starting Point for Adnectin Design.......................................6
4	Protein Engineering...................................................................7
4.1	Pharmaceutical Protein Formulations...................................................8
4.2	Improvements of Protein Stability and Solubility......................................8
4.3	The Incorporation of Disulfide Bonds into Engineered Proteins.........................9
4.3.1	Disulfide Bonds in Proteins...........................................................9
4.3.2	Disulfide Engineering................................................................10
4.3.3	Computational Methods for Disulfide Engineering......................................11
4.3.4	Disulfide by Design 2.0..............................................................12
CHAPTER 2 MATERIALS AND METHODS..............................................................15
1	Preparation of Recombinant Fn and their Mutants......................................15
1.1	Construction of Fn Mutants...........................................................15
1.1.1	List of Host Cell, Vector, and Culture Medium Recipes................................15
1.1.2	Construction of E1, E4 and their Variants Using FastCloning..........................16
1.1.3	Transformation of Plasmid into E. coli Strain XL1-blue...............................17
1.1.4	Transformation of Linearized Plasmid into E. coli Strain BL21 and Shuffle............17
2	Protein Expression of Fn Mutants.....................................................18
2.1	List of Culture Media and Solution Recipes...........................................18
2.2	Small Scale Protein Expression of Fn Mutants.........................................18
2.3	Large Scale Protein Expression of Fn Mutants.........................................18
3	Purification of Fn Mutants...........................................................19
3.1	List of Buffer Recipes...............................................................19
3.2	Purification of Fn Mutants by Ni2+-Chelating Chromatography..........................19
3.3	Purification of Fn Mutants by Reverse-Phase High Performance Liquid Chromatography	.............................................................................................19
4	Mass Spectrometric Measurement.......................................................20
5	Flow Cytometry.......................................................................20
5.1	List of Cell Line and Culture Medium.................................................20
5.2	List of Working Buffer and Reagent...................................................20
6	Cell Migration Assay.................................................................21
7	Differential Scanning Calorimetry (DSC)..............................................22
7.1.	List of DSC Buffer Recipes...........................................................22
7.2.	Thermostability Analysis of E1, E4 and their Variants in DSC.........................22
8	Prediction of Disulfide Bonds in E1, E4 by DbD2......................................23
CHAPTER 3 RESULTS............................................................................25
1	Inserting Disulfide Bonds in E1 and E4 by Bioinformatics Tool DbD2...................25
2	Construction of C-terminal Tail......................................................25
3	Expression, Purification and Mass Characterization of Fibronectin Mutants............25
4	Thermostability Analyzed by DSC......................................................26
5	The Solubility Analysis of E1, E4 and their Variants.................................27
6	The Effect of theirs Variant on EGFR Binding Affinity................................27
6.1	Inhibition of A549 Migration by E1, E4 and their Variants............................27
6.2	The Binding Affinity of EGFR to E1, E4 and their Variants Using Flow Cytometry.......28
CHAPTER 4 DISCUSSION.........................................................................29
1.	The Relationship Between Thermostability, Solubility and Binding Affinity............29
2.	The Differences Between the Inhibition of A549 Migration by E1, E4 and their Variants	.............................................................................................29
3.	The Differences Between the Inhibition of A549 Migration and the Binding of EGFR to E1, E4 and their Variants........................................................................30
4.	Introduction of Disulfide Bond Between Loop-L9 and β Strand-S95 can Improve the Stability, Solubility and Binding Affinity of Fn3 Scaffold...................................30
5.	The affection of C-terminal Addition of 7 Amino-acids on E4..........................31
CHAPTER 5 CONCLUSIONS........................................................................33
CHAPTER 6 FUTURE PERSPECTIVE.................................................................35
1.	As Alternative Therapy for Oncology and EGFR-Related Diseases........................35
2.	Lower Immunogenicity for Fibronectin Type III Domain.................................35
3.	Dual-Target Marker for Anti- EGFR....................................................35
REFERENCES...................................................................................37
RECIPES......................................................................................45
TABLES.......................................................................................50
FIGURES......................................................................................59
APPENDIX FIGURES.............................................................................80

                                    

Andrews, B.A., Schmidt, A.S., and Asenjo, J.A. Correlation for the partition behavior of proteins in aqueous two-phase systems: Effect of surface hydrophobicity and charge. Biotechnology and Bioengineering 90, 380–390.
Bagby, S., Tong, K.I., and Ikura, M. (2001). Optimization of protein solubility and stability for protein nuclear magnetic resonance. Meth. Enzymol. 339, 20–41.
Batori, V., Koide, A., and Koide, S. (2002). Exploring the potential of the monobody scaffold: effects of loop elongation on the stability of a fibronectin type III domain. Protein Eng. 15, 1015–1020.
Betz, S.F. (1993). Disulfide bonds and the stability of globular proteins. Protein Sci 2, 1551–1558.
Biggar, P., and Kim, G.-H. (2017). Treatment of renal anemia: Erythropoiesis stimulating agents and beyond. Kidney Res Clin Pract 36, 209–223.
Billings, K.S., Best, R.B., Rutherford, T.J., and Clarke, J. (2008). Crosstalk between the Protein Surface and Hydrophobic Core in a Core-swapped Fibronectin Type III Domain. J Mol Biol 375, 560–571.
Bloom, L., and Calabro, V. (2009). FN3: a new protein scaffold reaches the clinic. Drug Discovery Today 14, 949–955.
Bork, P., and Doolittle, R.F. (1992). Proposed acquisition of an animal protein domain by bacteria. Proc Natl Acad Sci U S A 89, 8990–8994.
Cao, Y., Liao, C., Tan, A., Liu, L., and Gao, F. (2010a). Meta-Analysis of Incidence and Risk of Hypomagnesemia with Cetuximab for Advanced Cancer. CHE 56, 459–465.
Cao, Y., Liu, L., Liao, C., Tan, A., and Gao, F. (2010b). Meta-analysis of incidence and risk of hypokalemia with cetuximab-based therapy for advanced cancer. Cancer Chemother. Pharmacol. 66, 37–42.
Carney, D.N., and Hansen, H.H. (2000). Non–Small-Cell Lung Cancer — Stalemate or Progress? New England Journal of Medicine 343, 1261–1262.
CHEN, P., WANG, L., LI, H., LIU, B., and ZOU, Z. (2013). Incidence and risk of hypomagnesemia in advanced cancer patients treated with cetuximab: A meta-analysis. Oncol Lett 5, 1915–1920.
Cho, J., and Mosher, D.F. Characterization of fibronectin assembly by platelets adherent to adsorbed laminin-111. Journal of Thrombosis and Haemostasis 4, 943–951.
Cota, E., Hamill, S.J., Fowler, S.B., and Clarke, J. (2000). Two proteins with the same structure respond very differently to mutation: the role of plasticity in protein stability11Edited by J. Thornton. Journal of Molecular Biology 302, 713–725.
Cota, E., Steward, A., Fowler, S.B., and Clarke, J. (2001). The folding nucleus of a fibronectin type III domain is composed of core residues of the immunoglobulin-like fold11Edited by A. R. Fersht. Journal of Molecular Biology 305, 1185–1194.
Couchman, J.R., Austria, M.R., and Woods, A. (1990). Fibronectin-Cell Interactions. Journal of Investigative Dermatology 94, s7–s14.
Craig, D.B., and Dombkowski, A.A. (2013). Disulfide by Design 2.0: a web-based tool for disulfide engineering in proteins. BMC Bioinformatics 14, 346.
Dani, V.S., Ramakrishnan, C., and Varadarajan, R. (2003). MODIP revisited: re-evaluation and refinement of an automated procedure for modeling of disulfide bonds in proteins. Protein Eng Des Sel 16, 187–193.
Demarest, S.J., Rogers, J., and Hansen, G. (2004). Optimization of the antibody C(H)3 domain by residue frequency analysis of IgG sequences. J Mol Biol 335, 41–48.
Demeule, B., Lawrence, M.J., Drake, A.F., Gurny, R., and Arvinte, T. (2007). Characterization of protein aggregation: the case of a therapeutic immunoglobulin. Biochim. Biophys. Acta 1774, 146–153.
Dickinson, C.D., Veerapandian, B., Dai, X.P., Hamlin, R.C., Xuong, N.H., Ruoslahti, E., and Ely, K.R. (1994). Crystal structure of the tenth type III cell adhesion module of human fibronectin. J. Mol. Biol. 236, 1079–1092.
Dobson, C.M. (2003). Protein folding and misfolding. Nature 426, 884–890.
Dombkowski, A.A. (2003). Disulfide by Design: a computational method for the rational design of disulfide bonds in proteins. Bioinformatics 19, 1852–1853.
Dombkowski, A.A., and Crippen, G.M. (2000). Disulfide recognition in an optimized threading potential. Protein Eng Des Sel 13, 679–689.
Dombkowski, A.A., Sultana, K.Z., and Craig, D.B. (2014). Protein disulfide engineering. FEBS Lett. 588, 206–212.
Emanuel, S.L., Engle, L.J., Chao, G., Zhu, R.-R., Cao, C., Lin, Z., Yamniuk, A., Hosbach, J., Brown, J., Fitzpatrick, E., et al. (2011). A fibronectin scaffold approach to bispecific inhibitors of epidermal growth factor receptor and insulin-like growth factor-I receptor. MAbs 3, 38–48.
Famm, K., and Winter, G. (2006). Engineering aggregation-resistant proteins by directed evolution. Protein Eng. Des. Sel. 19, 479–481.
Fowler, S.B., Poon, S., Muff, R., Chiti, F., Dobson, C.M., and Zurdo, J. (2005). Rational design of aggregation-resistant bioactive peptides: Reengineering human calcitonin. Proc Natl Acad Sci U S A 102, 10105–10110.
Frokjaer, S., and Otzen, D.E. (2005). Protein drug stability: a formulation challenge. Nature Reviews Drug Discovery 4, 298–306.
Gidalevitz, T., Stevens, F., and Argon, Y. (2013). Orchestration of secretory protein folding by ER chaperones. Biochim Biophys Acta 1833, 2410–2424.
Gill, P., Moghadam, T.T., and Ranjbar, B. (2010). Differential Scanning Calorimetry Techniques: Applications in Biology and Nanoscience. J Biomol Tech 21, 167–193.
Goldstraw, P., Chansky, K., Crowley, J., Rami-Porta, R., Asamura, H., Eberhardt, W.E.E., Nicholson, A.G., Groome, P., Mitchell, A., Bolejack, V., et al. (2016). The IASLC Lung Cancer Staging Project: Proposals for Revision of the TNM Stage Groupings in the Forthcoming (Eighth) Edition of the TNM Classification for Lung Cancer. Journal of Thoracic Oncology 11, 39–51.
Haynie, D.T., and Freire, E. (1994). Estimation of the folding/unfolding energetics of marginally stable proteins using differential scanning calorimetry. Anal. Biochem. 216, 33–41.
Hazes, B., and Dijkstra, B.W. (1988). Model building of disulfide bonds in proteins with known three-dimensional structure. Protein Eng. 2, 119–125.
Huveneers, S., and Danen, E.H.J. (2009). Adhesion signaling – crosstalk between integrins, Src and Rho. Journal of Cell Science 122, 1059–1069.
Hynes, R.O., Schwarzbauer, J.E., and Tamkun, J.W. (1984). Fibronectin: a versatile gene for a versatile protein. Ciba Found. Symp. 108, 75–92.
Jacobs, S.A., Wu, S.-J., Feng, Y., Bethea, D., and O’Neil, K.T. (2010). Cross-interaction chromatography: a rapid method to identify highly soluble monoclonal antibody candidates. Pharm. Res. 27, 65–71.
Jean E. Schwarzbauer. (1991). Fibronectin: from gene to protein. Current Opinion in Cell Biology 1991, 3:786-791.
Kapila, Y.L., Wang, S., Dazin, P., Tafolla, E., and Mass, M.J. (2002). The Heparin-binding Domain and V Region of Fibronectin Regulate Apoptosis by Suppression of p53 and c-myc in Human Primary Cells. J. Biol. Chem. 277, 8482–8491.
Katzel, J.A., Fanucchi, M.P., and Li, Z. (2009). Recent advances of novel targeted therapy in non-small cell lung cancer. J Hematol Oncol 2, 2.
Kerns, E.H., and Di, L. (2003). Pharmaceutical profiling in drug discovery. Drug Discov. Today 8, 316–323.
Kim, D.Y., Kandalaft, H., Ding, W., Ryan, S., van Faassen, H., Hirama, T., Foote, S.J., MacKenzie, R., and Tanha, J. (2012). Disulfide linkage engineering for improving biophysical properties of human VH domains. Protein Eng. Des. Sel. 25, 581–589.
Kobayashi, S., Boggon, T.J., Dayaram, T., Jänne, P.A., Kocher, O., Meyerson, M., Johnson, B.E., Eck, M.J., Tenen, D.G., and Halmos, B. (2009). EGFR Mutation and Resistance of Non–Small-Cell Lung Cancer to Gefitinib.
Koide, A., and Koide, S. (2007). Monobodies. In Protein Engineering Protocols, (Humana Press), pp. 95–109.
Koide, A., Bailey, C.W., Huang, X., and Koide, S. (1998). The fibronectin type III domain as a scaffold for novel binding proteins11Edited by J. Wells. Journal of Molecular Biology 284, 1141–1151.
Koide, A., Gilbreth, R.N., Esaki, K., Tereshko, V., and Koide, S. (2007). High-affinity single-domain binding proteins with a binary-code interface. PNAS 104, 6632–6637.
Kragelund, B.B., Jönsson, M., Bifulco, G., Chazin, W.J., Nilsson, H., Finn, B.E., and Linse, S. (1998). Hydrophobic core substitutions in calbindin D9k: effects on Ca2+ binding and dissociation. Biochemistry 37, 8926–8937.
Krishnamurthy, R., and Manning, M. (2002). The Stability Factor: Importance in Formulation Development. Current Pharmaceutical Biotechnology 3, 361–371.
Le, Q.A.T., Joo, J.C., Yoo, Y.J., and Kim, Y.H. (2012). Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge. Biotechnol. Bioeng. 109, 867–876.
Lee, E.N., Kim, Y.M., Lee, H.J., Park, S.W., Jung, H.Y., Lee, J.M., Ahn, Y.-H., and Kim, J. (2005). Stabilizing peptide fusion for solving the stability and solubility problems of therapeutic proteins. Pharm. Res. 22, 1735–1746.
Lienqueo, M.E., Mahn, A., Navarro, G., Salgado, J.C., Perez‐Acle, T., Rapaport, I., and Asenjo, J.A. New approaches for predicting protein retention time in hydrophobic interaction chromatography. Journal of Molecular Recognition 19, 260–269.
Lipovšek, D. (2011). Adnectins: engineered target-binding protein therapeutics. Protein Eng Des Sel 24, 3–9.
Liu, T.-C., Jin, X., Wang, Y., and Wang, K. (2017a). Role of epidermal growth factor receptor in lung cancer and targeted therapies. Am J Cancer Res 7, 187–202.
Liu, X., Wang, P., Zhang, C., Ma, Z., Liu, X., Wang, P., Zhang, C., and Ma, Z. (2017b). Epidermal growth factor receptor (EGFR): A rising star in the era of precision medicine of lung cancer. Oncotarget 8, 50209–50220.
Lobstein, J., Emrich, C.A., Jeans, C., Faulkner, M., Riggs, P., and Berkmen, M. (2012). SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microbial Cell Factories 11, 753.
Main, A.L., Harvey, T.S., Baron, M., Boyd, J., and Campbell, I.D. (1992). The three-dimensional structure of the tenth type III module of fibronectin: An insight into RGD-mediated interactions. Cell 71, 671–678.
Mao, Y., and Schwarzbauer, J.E. (2005). Fibronectin fibrillogenesis, a cell-mediated matrix assembly process. Matrix Biology 24, 389–399.
Marrink, J., vd Geest, S., Sinnema, R., Vellenga, E., and de Vries, E.G. (1985). Fibronectin-complex (FN-C) present in normal plasma. Thromb. Res. 37, 689–692.
Mårtensson, L.-G., Karlsson, M., and Carlsson, U. (2002). Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond. Biochemistry 41, 15867–15875.
Matsumura, M., Becktel, W.J., Levitt, M., and Matthews, B.W. (1989a). Stabilization of phage T4 lysozyme by engineered disulfide bonds. Proc Natl Acad Sci U S A 86, 6562–6566.
Matsumura, M., Signor, G., and Matthews, B.W. (1989b). Substantial increase of protein stability by multiple disulphide bonds. Nature 342, 291–293.
Melnik, B.S., Povarnitsyna, T.V., Glukhov, A.S., Melnik, T.N., Uversky, V.N., and Sarma, R.H. (2012). SS-Stabilizing Proteins Rationally: Intrinsic Disorder-Based Design of Stabilizing Disulphide Bridges in GFP. J. Biomol. Struct. Dyn. 29, 815–824.
Muro, A.F., Caputi, M., Pariyarath, R., Pagani, F., Buratti, E., and Baralle, F.E. (1999). Regulation of Fibronectin EDA Exon Alternative Splicing: Possible Role of RNA Secondary Structure for Enhancer Display. Mol Cell Biol 19, 2657–2671.

Neira, J.L., Davis, B., Ladurner, A.G., Buckle, A.M., Gay, P.G., and Fersht, A.R. (1996). Towards the complete structural characterization of a protein folding pathway: the structures of the denatured, transition and native states for the association/folding of two complementary fragments of cleaved chymotrypsin inhibitor 2. Direct evidence for a nucleation-condensation mechanism. Fold Des 1, 189–208.
Normanno, N., De Luca, A., Bianco, C., Strizzi, L., Mancino, M., Maiello, M.R., Carotenuto, A., De Feo, G., Caponigro, F., and Salomon, D.S. (2006). Epidermal growth factor receptor (EGFR) signaling in cancer. Gene 366, 2–16.
Pawar, A.P., Dubay, K.F., Zurdo, J., Chiti, F., Vendruscolo, M., and Dobson, C.M. (2005). Prediction of “aggregation-prone” and “aggregation-susceptible” regions in proteins associated with neurodegenerative diseases. J. Mol. Biol. 350, 379–392.
Perchiacca, J.M., Ladiwala, A.R.A., Bhattacharya, M., and Tessier, P.M. (2012). Aggregation-resistant domain antibodies engineered with charged mutations near the edges of the complementarity-determining regions. Protein Eng Des Sel 25, 591–602.
Petersen, M.T., Jonson, P.H., and Petersen, S.B. (1999). Amino acid neighbours and detailed conformational analysis of cysteines in proteins. Protein Eng. 12, 535–548.
Plaxco, K.W., Spitzfaden, C., Campbell, I.D., and Dobson, C.M. (1997). A comparison of the folding kinetics and thermodynamics of two homologous fibronectin type III modules11Edited by P. E. Wright. Journal of Molecular Biology 270, 763–770.

Ramamurthy, V., Krystek Jr., S.R., Bush, A., Wei, A., Emanuel, S.L., Das Gupta, R., Janjua, A., Cheng, L., Murdock, M., Abramczyk, B., et al. (2012). Structures of Adnectin/Protein Complexes Reveal an Expanded Binding Footprint. Structure 20, 259–269.
Robert, F., Blumenschein, G., Herbst, R.S., Fossella, F.V., Tseng, J., Saleh, M.N., and Needle, M. (2005). Phase I/IIa Study of Cetuximab With Gemcitabine Plus Carboplatin in Patients With Chemotherapy-Naïve Advanced Non–Small-Cell Lung Cancer. JCO 23, 9089–9096.
Rozners, E., Pilch, D.S., and Egli, M. (2015). Calorimetry of Nucleic Acids. Curr Protoc Nucleic Acid Chem 63, 7.4.1-12.
Ruoslahti, E. (1984). Fibronectin in cell adhesion and invasion. Cancer Metastasis Rev. 3, 43–51.
Sasaki, T., Hiroki, K., and Yamashita, Y. (2013). The Role of Epidermal Growth Factor Receptor in Cancer Metastasis and Microenvironment. Biomed Res Int 2013.
Sharma, A., Askari, J.A., Humphries, M.J., Jones, E.Y., and Stuart, D.I. (1999). Crystal structure of a heparin- and integrin-binding segment of human fibronectin. EMBO J 18, 1468–1479.
Siadat, O.R., Lougarre, A., Lamouroux, L., Ladurantie, C., and Fournier, D. (2006). The effect of engineered disulfide bonds on the stability of Drosophila melanogaster acetylcholinesterase. BMC Biochem 7, 12.
Siggers, K., Soto, C., and Palmer, A.G. (2007). Conformational Dynamics in Loop Swap Mutants of Homologous Fibronectin Type III Domains. Biophys J 93, 2447–2456.
Sowdhamini, R., Srinivasan, N., Shoichet, B., Santi, D.V., Ramakrishnan, C., and Balaram, P. (1989). Stereochemical modeling of disulfide bridges. Criteria for introduction into proteins by site-directed mutagenesis. Protein Eng. 3, 95–103.
Su, X., Lacouture, M.E., Jia, Y., and Wu, S. (2009). Risk of High-Grade Skin Rash in Cancer Patients Treated with Cetuximab – an Antibody against Epidermal Growth Factor Receptor: Systemic Review and Meta-Analysis. OCL 77, 124–133.
Swencki-Underwood, B., Mills, J.K., Vennarini, J., Boakye, K., Luo, J., Pomerantz, S., Cunningham, M.R., Farrell, F.X., Naso, M.F., and Amegadzie, B. (2008). Expression and characterization of a human BMP-7 variant with improved biochemical properties. Protein Expression and Purification 57, 312–319.
Teilum, K., Olsen, J.G., and Kragelund, B.B. (2011). Protein stability, flexibility and function. Biochim. Biophys. Acta 1814, 969–976.
Thornton, J.M. (1981). Disulphide bridges in globular proteins. Journal of Molecular Biology 151, 261–287.
Tischenko, V.M., Abramov, V.M., and Zav’yalov, V.P. (1998). Investigation of the cooperative structure of Fc fragments from myeloma immunoglobulin G. Biochemistry 37, 5576–5581.
Trevino, S.R., Scholtz, J.M., and Pace, C.N. (2007). Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa. J. Mol. Biol. 366, 449–460.
Uramoto, H., and Tanaka, F. (2014). Recurrence after surgery in patients with NSCLC. Transl Lung Cancer Res 3, 242–249.
Veronese, F.M., and Mero, A. (2008). The Impact of PEGylation on Biological Therapies. BioDrugs 22, 315–329.
Wahlberg, E., and Härd, T. (2006). Conformational Stabilization of an Engineered Binding Protein. J. Am. Chem. Soc. 128, 7651–7660.
Wedekind, J. (1988). Computer-Aided Model Building. In Learning Issues for Intelligent Tutoring Systems, (Springer, New York, NY), pp. 287–294.
Williams, E.C., Janmey, P.A., Johnson, R.B., and Mosher, D.F. (1983a). Fibronectin. Effect of disulfide bond reduction on its physical and functional properties. J. Biol. Chem. 258, 5911–5914.
Williams, E.C., Janmey, P.A., Johnson, R.B., and Mosher, D.F. (1983b). Fibronectin. Effect of disulfide bond reduction on its physical and functional properties. J. Biol. Chem. 258, 5911–5914.
Wu, S.-J., Luo, J., O’Neil, K.T., Kang, J., Lacy, E.R., Canziani, G., Baker, A., Huang, M., Tang, Q.M., Raju, T.S., et al. (2010). Structure-based engineering of a monoclonal antibody for improved solubility. Protein Eng Des Sel 23, 643–651.
Xu, L., Aha, P., Gu, K., Kuimelis, R.G., Kurz, M., Lam, T., Lim, A.C., Liu, H., Lohse, P.A., Sun, L., et al. (2002). Directed Evolution of High-Affinity Antibody Mimics Using mRNA Display. Chemistry & Biology 9, 933–942.
Yazdi, M.H., Faramarzi, M.A., Nikfar, S., and Abdollahi, M. (2015). A Comprehensive Review of Clinical Trials on EGFR Inhibitors Such as Cetuximab and Panitumumab as Monotherapy and in Combination for Treatment of Metastatic Colorectal Cancer. Avicenna J Med Biotechnol 7, 134–144.
Yun, C.-H., Mengwasser, K.E., Toms, A.V., Woo, M.S., Greulich, H., Wong, K.-K., Meyerson, M., and Eck, M.J. (2008). The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci U S A 105, 2070–2075.
Zakrzewska, M., Krowarsch, D., Wiedlocha, A., Olsnes, S., and Otlewski, J. (2005). Highly stable mutants of human fibroblast growth factor-1 exhibit prolonged biological action. J. Mol. Biol. 352, 860–875.
US Patent Application for METHODS FOR MAINTAINING PEGYLATION OF POLYPEPTIDES Patent Application (Application #20170049905 issued February 23, 2017) - Justia Patents Search.
張永昇 (2013). Design, structure determination, and biological evaluation of potent integrin α5β1 and/or αvbβ3-specific antagonist using the ninth and/or tenth module of fibronectin type III domain. Doctor Degree, National Cheng Kung University, Taiwan.

陳昰全 (2014). Development of dual VEGFR2- and integrin α5β1-specific antagonist using the fibronectin type III domain. Master Degree, National Cheng Kung University, Taiwan.

林舉征 (2017). Protein engineering of VEGFR2-specific antagonist using the fibronectin type III domain to improve its thermostability, and solubility. Master Degree, National Cheng Kung University, Taiwan.

校外：不公開電子論文及紙本論文均尚未授權公開

簡易檢索 / 詳目顯示

相關論文