陳長謙臺灣大學：化學研究所林素卿Lin, Su-ChingSu-ChingLin2007-11-262018-07-102007-11-262018-07-102007http://ntur.lib.ntu.edu.tw//handle/246246/51880膜蛋白在生物系統中飾演著非常基本且重要的角色，它的主要功能為： 細胞表面物質之受質(receptors)，離子通道(ion channels)，穿透膜之運輸子 (trans-membrane transports)， 訊息傳遞者(signal transducers)，離子幫浦(ion pumps) 及能量運輸子(free energy transducers)。本論文有興趣於兩種膜蛋白系統：膜接合 孕酮受質蛋白(membrane-associated progesterone receptors；MAPRs)與微粒體甲烷 單氧酵素(particulate methane monooxygenase；pMMO)。 其中發生在膜接合孕酮受質蛋白(MAPRs)的賀爾蒙具有促使非基因性且 快速訊息傳遞之行為。我們成功地運用sticky-end 聚合酶反應將人類肝中基因轉 殖到大腸桿菌，經大量繁殖與蛋白質表現及金屬離子親和層析法(metal-affinity chromatography)純化後，可有效地獲得兩個具有His-tag 及GST 融合的重組型膜 接合孕酮受質蛋白(pET21mapr 和pGEX_mapr)。藉由一系列光譜分析和分辨分子 大小層析法(Size Exclusion Chromatography)得知：重組膜接合孕酮受質蛋白 pET21mapr，無論於模擬生物膜的界面活性劑環境下與否，皆具有明顯α-helical 結 構；於模擬生物膜的界面活性劑環境時，由於親油性作用下促使此膜蛋白聚合分 子量大為減小，如此也可減緩膜蛋白聚集沉積。它也是一種具有高自旋氧化態鐵 離子(high spin Fe (III))鍵結的紫質鍵結蛋白(heme-binding protein)。賀爾蒙(孕酮) 的作用可以穩定pET21mapr 的結構，避免受孕酮攜帶分子-有機溶劑(如DMSO) VI 破壞。於核磁共振2D TROSY-based 1H-15N HSQC 實驗結果可推知，孕酮可能作 用於重組膜接合孕酮受質蛋白pET21mapr 的蛋白氨酸(arginine)及氨基乙酸 (glycine) 等殘基上。 另一類頗感興趣的膜蛋白系統為微粒體甲烷單氧酵素pMMO，它由三個 次單元體組成，其中最為重要者為具有N-端及C-端水曝露區的pmob 次單元體， 它可能是電子傳遞銅離子簇(E-cluster)所在，用於提供電子給基質催化離子簇 (C-cluster)進行甲烷的氫氧化反應。我們成功地藉由分子生物技術方法而獲得一系 列重組pmoB 蛋白；並由銅離子之鍵結研究發現C-端水曝露區的pmob 次單元體 pmoBCW 蛋白可與10 個以上還原態銅離子(Cu(I))鍵結，雖然解離常數為330 μM，但有高達4.3 互助性(cooperactivity)的Hill 常數，此性質正意謂著銅離子易 於純化過程中遺漏，因此含有不足量銅離子之x-ray 繞射晶體pMMO 酵素總缺少 活性。本論文研究結果，正可瞭解還原態電子傳遞銅離子簇於pMMO 酵素催化反 應中所扮演的重要角色。Membrane proteins play significant roles in biological systems and function as receptors, ionic channels, trans-membrane transporters, signal transducers, ion pumps and free energy transducers, etc. In this dissertation, we focus on two membranes proteins, membrane-associated progesterone receptors (MAPRs) and particulate methane monooxygenase (pMMO). Steroid hormones can mediate rapid non-genomic effects by binding to membrane-associated receptors. We have utilized the strategy of sticky-end PCR for efficient cloning of the human liver MAPR in E. coli. BL21 (DE3) strain, successfully purified and obtained two recombinant proteins, dubbed pET21mapr and pGEX_mapr by using metal-affinity or/and glutathione S-transferase affinity column. From spectroscopic studies and size-exclusion chromatography analysis, the following results were obtained. The recombinant protein pET21mapr exhibits α-helical structure both in the presence or absence of detergents. There is strong hydrophobic interaction between detergent n-dodecyl-β-maltoside and membrane-bound protein. The interaction could be used to decrease the oligomeric state of proteins and alleviate protein aggregation. In CD studies, we show that the hydrophobic steroid hormone progesterone protect the secondary structure of progesterone receptor in the absence of detergent against the organic solvent DMSO, which is typically used to deliver progesterone to the receptor. From 2D TROSY-based VIII 1H-15N HSQC spectra, it appears that progesterone binds to the pET21mapr receptor at residues of arginine, glycine and others. More NMR experiments are required before the extract residues could be determined. A second membrane protein is the pMMO enzyme from Methylococcus capsulatus which consists of three subunits named pmoA, pmoB and pmoC. Subunit pmoB is the largest one, and possesses two large N-terminal and C-terminal water-exposed domains, which serve as the location of the E-clusters to provide a reservoir of reducing equivalents for re-reduction of the C-clusters following methane hydroxylation. In this dissertation, these recombinant pmoB proteins have to successfully been cloned, expressed and purified. From Scatchard plots of copper binding, we have concluded that the C-terminus water-exposed pmoBCW domain can accommodate more than ten Cu(I) ions with dissociation constant 330μM , and that the Cu(I) binding is highly cooperative (αH=4.3). These results clarify why the copper ions are so readily lost during the purification process. In the x-ray structural analysis, the preparation was devoid of ~10 copper ions, and did not possess biochemical activity. In short, the preparation lacked the full complement of copper ions to be functional. Thus, we have contributed to the understanding of the significance and the role of the reduced E-cluster in the turnover of the pMMO.Reconstitution of Membrane Proteins or Subunits for Structural and Functional Studies Acknowledgement (Chinese) Abstract (Chinese) Abstract (English) Introduction Part I: Membrane Associated Progesterone Receptors (MAPRs) for structural and Functional Studies Chapter 1 Introduction 1.1 Two mechanisms of steroid actions 1.1.1 Genomic Action of steroid hormones∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙5 1.1.2 Nongenomic steroid action∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙6 1.2 The structure of human intracellular progesterone receptor for ligand binding domain (hPR LBD) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙12 1.3 The prediction of the structure of Membrane associated Progesterone Receptors (MAPRs) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙15 Chapter 2 experimental methods 2.1 Materials and instruments 2.1.1 Materials ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙18 2.1.2 Instruments ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙22 2.2 Construction of expression vectors by using sticky-end method 2.2.1 Target genes preparation: Amplification, purification and phosphorylation∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙24 2.2.2 Vectors preparation: Plasmid extraction, restriction enzyme digestion, dephosphorylation and purification∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙29 X 2.2.3 Ligation and transformation of recombinant genes∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙31 2.2.4 Recombinants screening and protein verification∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙33 2.3 Over-expression and purification of recombinant proteins∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙34 Chapter 3 Results and Discussion 3.1 Construction of expression vectors by sticky-end method 3.1.1 Target genes preparation: Amplification, Purifiction, and Phosphorylation∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙41 3.1.2 Vector preparation, ligation reaction and recombinant screen∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙44 3.2 Harvesting, extraction and purification of recombinant proteins 3.2.1 Harvesting and extraction of recombinant proteins∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙54 3.2.2 Purification of recombinant pGEX_mapr and pET21mapr proteins∙∙∙∙∙∙∙∙∙∙∙∙57 3.2.3 Determination of the Oligomeric State∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙62 3.3 Characterization by CD, UV, Fluorescence, EPR and NMR spectroscopies∙∙∙∙∙∙∙∙∙∙∙64 Chapter 4 Conclusion ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙76 Reference ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙79 Appendix ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙83 XI Part II: Particulate Methane Monooxygenase (pMMO) for Structural and Functional Studies Chapter 1 Introduction 1.1 The importances of methanotrophs ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙89 1.2 The role of copper in pMMO system ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙94 1.3 The structure of pmoB : E-cluster complex 1.3.1 Rosenzweig’s crystal structure∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙100 1.3.2 Chan’s predicted structure∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙100 1.4 Goals of the work described in the part of the dissertation ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙103 Chapter 2 Methods and experiments 2.1 Methods ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙104 2.2 Experiments 2.2.1 Bacterial strains, plasmids and growth conditions ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙106 2.2.2 Preparation of genomic DNA from Methylococcus capsulatus (Bath) ∙∙∙∙∙∙106 2.2.3 Insertion of pMMO related gene to appropriate overexpression vectors in E. coli ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙107 2.2.4 Over-expressed proteins induction in E. coli BL21 (DE3) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙107 2.2.5 SDS_PAGE analysis of small scale over-expressed protein ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙109 2.2.6 Purification of recombinant DNA over-expressed proteins ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙109 Chapter 3 Results and Discussion 3.1 Over-expression of the N-terminus and C-terminus sub-domains of the pmoB subunit-fusion with poly-his-tag in E. Col ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙111 3.2 The affinity of reduced cupric ions toward the pmoBCW1 V322E E343D sub-domain ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙113 3.3 Affinity studies of the reduction of cupric Ions toward the GST-fused pmoBCW sub-domain ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙118 XII 3.4 Affinity studies of the reduction of cupric Ions toward the Nus-tagged pmoBCW+TM sub-domain ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙119 Chapter 4 Conclusions ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙122 References ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙127 XIII Part I: Membrane Associated Progesterone Receptors (MAPRs) For Structural and Functional Studies Figure (A). Trans-membrane proteins in the phospholipids bilayer: a cartoon ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙3 Figure 1-1. General structural and functional organization of intracellular steroid hormone receptors ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙10 Figure 1-2. Activation of a steroid hormone receptor ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙10 Figure 1-3. Schematic presentation of the two-step model for steroid action comprising genomic and nongenomic actions of steroids exemplified for aldosterone ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙11 Figure 1-4. (A) Ribbons diagram of the liganded hPR LBD (B) Comparison of The PR LBD and RXR LBD dimmer ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙14 Figure 1-5 (A) represent interactions between the PR LBD and bound progesterone (B) the structure of anti-progestin, RU486 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙14 Figure 1-6. Structure of the heme-binding domain in bovine cytochrome b5 ∙∙∙∙∙∙∙∙∙17 Figure 2-1. Separation of proteins in an 8.3 ml bed volume of PD-10 desalting column ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙38 Figure 3-1. Molecular cloning strategy by the sticky-end method ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙43 Figure 3-3. Recombinant plasmids of pGEX_Mapr after ligation reaction and incubation for overnight at 37℃ were screened by EcoRI and XhoI digestions and analyzed by 0.8% agarose gel electrophoresis ∙∙∙∙∙∙∙∙∙∙∙∙∙∙47 Figure 3-4 Recombinant plasmids of pGEX Mapr after ligation reaction were screened by PCRs and analyzed by 2% agarose gel electrophoresis ∙∙∙∙47 Figure 3-5. DNA sequence alignment between pGEX_mapr and PGRMC1 (Homo sapiens progesterone receptor membrane component 1) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙48 Figure 3-6. Protein sequence alignment between pGEX_mapr and PGRMC1 ∙∙∙∙∙∙∙50 Figure 3-7. DNA sequence alignment between pET21Mapr and PGRMC1(Homo sapiens progesterone receptor membrane component 1) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙511830610 bytesapplication/pdfen-US膜接合孕酮受質蛋白賀爾蒙紫質鍵結蛋白微粒體甲烷單氧酵素MAPRshormoneheme-binding proteinpMMOE-cluster and C-clusterthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/51880/1/ntu-96-D88223018-1.pdf