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研究生:白莉敏
研究生(外文):Prabha Regmi
論文名稱:以桿狀病毒系統表現野生型與突變型高胱胺酸及其特性分析之研究
論文名稱(外文):Expression and Characterization of Wild type and MutantCystathionine-beta Synthase in Baculovirus Expression System
指導教授:吳宗遠
指導教授(外文):Tzong Yuan Wu
學位類別:碩士
校院名稱:中原大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:英文
論文頁數:92
中文關鍵詞:胱硫醚合酶半胱胺酸突變
外文關鍵詞:homocysteinemutationCystathionine beta-synthase
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胱硫醚貝他合成酵素(cystathionine-β-synthase, CBS)可經由轉硫作用 (transsulfuration)可將高半胱胺酸(homocysteine)合成胱胺酸(cystine;Cys)的一個重要催化酵素,此過程為不可逆轉的反應。胱硫醚貝他合成酵素功能的缺乏會造成體內過度堆積高胱胺酸(methionine)而導致高胱胺酸血症 (hyperhomocysteinemia),且常伴隨心血管疾病,骨質疏鬆,骨骼畸型,眼球水晶體脫位,甚至可能造成神經病變。CBS的功能缺陷也是所有先天代謝缺陷中最普遍的類別之一,目前為止,已有130種CBS突變被文獻記載。
日前在台灣蘭嶼發現新的CBS錯義突變,且發生率高達百分之一。主要突變位置在CBS 蛋白質N端內一個鐵結合的區域 (heme-binding region) 之第四十七個胺基酸。此位置發生突變的高發生率突顯了意味著研究CBS酵素活性的重要性。因此本研究想利用桿狀病毒表現系統大量表現野生型CBS以及突變型 D47E (CBS) ,進而純化和分析CBS酵素活性的差異。為了加速純化的便利性,我們在CBS的N端加了六個histidine。可以由西方點墨法分析CBS蛋白質的表現量和純化結果。
由於利用桿狀病毒表現系統優勢,可以輕易的生產出大量的CBS蛋白,這有利於進一步研究突變型CBS蛋白的生化,生物物理特性,也更利於了解此病症的背後機轉。


Cystathionine beta-synthase (CBS) is a crucial enzyme that catalyzes the irreversible, serine-dependent conversion of homocysteine to cystathionine via a transsulfuration pathway. Deficiency of this enzyme leads to severe hyperhomocysteinemia along with diverse clinical manifestations like; cardiovascular disease, osteoporosis, skeletal abnormalities, dislocated optic lens and even neurological abnormalities.CBS deficiency is one of the most common types of inborn errors of metabolism and more than 130 mutations have been reported to date.
In context to Taiwan, a new missense mutation on CBS gene has been found in the population of Orchid Island with very high incidence of 1:100. The mutation is located in the 47th amino residues in the heme-binding region in N-terminal of the CBS protein. The high incidence in that population signifies the necessity of study on CBS enzyme. Therefore, our research focused on the overexpression of wild type and mutant CBS (D47E) in Baculovirus Expression Vector System (BEVS). The expression and purification of both the wild type and mutant CBS provide information on its enzyme activity which is a vital response in non-pathogenic state. To facilitate the purification 6X His was tagged in the N terminal of CBS. The expression and purification had been confirmed by Western Blot.
By BEVS, it is easy to produce large quantity of CBS protein which can facilitate on studying biochemical, biophysical properties and to better understand the pathological mechanism of the mutant CBS found in Taiwan.


TABLE OF CONTENTS
中文摘要...................................................................................................................... I
ABSTRACT ................................................................................................................ II
ACKNOWLEDGEMENT .........................................................................................III
TABLE OF CONTENTS ......................................................................................... IV
LIST OF TABLES ........................................................................................................ VII
LIST OF FIGURES ...................................................................................................... VIII
ABBREVIATIONS .................................................................................................. IX
DEDICATION .......................................................................................................... XI
1.1 Background........................................................................................................... 1
1.2 Problem Statement ............................................................................................... 2
1.3 Objective ............................................................................................................... 2
I. LITERATURE REVIEW ......................................................................................... 3
2.1 Homocysteine and its importance ....................................................................... 3
2.2 The metabolism of homocystetine: Pathway and Regulation .............................. 4
2.3 Role of Cystathionine-β-synthase in Transulfuration Pathway .......................... 6
2.4 Characterization of Cystathionine β-synthase .................................................... 7
2.4.1 Catalytic Regulation of Cystathioine ........................................................... 7
2.4.2 Regualtion of Cystathionine ......................................................................... 8
2.4.3 Relationship between CBS and associated enzyme: .................................... 8
2.4.4 Reactions catalyzed by CBS .......................................................................... 9
2.5 Cystathionine β-synthase (CBS) Gene ..............................................................11
2.6 CBS as a Modular protein .................................................................................12
2.6.1 Heme Binding Domain ..............................................................................13
2.6.2 Active core of CBS ........................................................................................14
2.6.3 Catalytic Domain .........................................................................................14
2.6.4 Regulatory domain .....................................................................................14
2.7 Crystal Structure ................................................................................................. 15
2.8 Kinetic studies ....................................................................................................17
2.9 CBS Mutation .....................................................................................................17
2.10 Expression and Purification of CBS..................................................................20
2.11 Expression System ............................................................................................20
2.11.1 Baculovirus Expression System ................................................................20
2.11.1.1 Introduction to baculovirus .........................................................................23
2.11.1.2 Enhancing the expression by Modifying baculovirus strains .....................24
2.12 Insect Cell Culture ..............................................................................................25
2.13 Internal Ribosome Entry Site (IRES) ................................................................. 25
2.14 Purification and Analysis of Enzyme Activity ...................................................26
2.15 Site Directed Mutagenesis ..................................................................................27
II. MATERIALS and Methods ....................................................................................29
3 Experimental Procedure .........................................................................................29
3.1 Cloning of the human CBS cDNA into Expression Vector ...............................29
3.2 Generation of CBS Mutant and Purification ......................................................30
3.3 Expression and Purification of recombinant CBS .............................................31
3.4 Methodology and Materials for Polymerase Chain Reaction ............................32
3.5 Methodology and Materials for Gel Electrophoresis .........................................34
3.6 Transformation ...................................................................................................35
3.7 Plasmid DNA Extraction ....................................................................................35
3.7.1 Mini-prep Plasmid Extraction...........................................................................35
3.7.2 Plasmid Extraction by High –Speed Plasmid Mini Kit ....................................36
3.7.3 Plasmid Extraction by Plasmid Midi prep-kit .............................................36
3.8 Competent Cell ...................................................................................................37
3.9 Cloning ...............................................................................................................37
3.10 DNA Gel Extraction ...........................................................................................39
3.11 Cell Culture ........................................................................................................39
3.12 Generating Recombinant Baculoviruses by Co-Transfection ............................40
3.13 End -point Dilution Assay ..................................................................................41
3.14 Amplying Virus ..................................................................................................42
3.15 Titre Determination ............................................................................................42
3.16 Protein Extraction ...............................................................................................43
3.17 Sodium dodecyl sulfate (SDS-PAGE) and Western Blot Analysis ...................44
3.18 Site directed mutagenesis ...................................................................................47
3.19 Analytical procedures .........................................................................................48
3.19.1 Protein Concentration .....................................................................................48
3.19.2 Spectroscopic Study ...................................................................................48
III. RESULTS ...............................................................................................................49
4.1 Construction and generation of Expression vectors ..........................................49
4.2 Expression of hCBS and mutant CBS in Sf21 cell ............................................50
4.3 Optimizing Gene Expression ..........................................................................50
4.4 Purification of CBS protein ................................................................................51
4.5 The CBS expressed in Insect cell does not have a heme group .........................51
4.6 Chemical crosslinking of CBS protein ...............................................................51
IV. CONCLUSION .......................................................................................................53
V. DISCUSSION .........................................................................................................54
VI. RECOMMENDATION ..........................................................................................58
VII. REFRENCES ..........................................................................................................60
LIST OF TABLES
Table 1: Optimized condition for human CBS cDNA Amplification .............32
Table 2: Optimized condition for Generation of Mutant plasmid ...................32
Table 3: Optimized condition for Generation of 6Xhis tagged CBS...............33
Table 4: Optimized condition for Generation of 6Xhis tagged mutantCBS ...33
Table 5: Number of cells in different flask and plate ......................................40
LIST OF FIGURES
Figure 1: Fates of Methionine and Homocysteine Metabolism ......................... 5
Figure 2: Modular domain structure of human CBS .......................................12
Figure 3: Crystal structure of the truncated dimeric form of CBS ..................16
Figure 4: The structure of the dimer of truncated human CBS .......................19
Figure 5: Ketoenamine and enolimine tautomers of internal aldimine of PLP
in hCBS (Weeks et al. 2009). ...................................................................55
Figure 6: UV-visible spectra of recombinant yeast CBS. Inset: formation of
PLP-hydroxylamine oxime ......................................................................57
Figure 7: Construction of Recombinant Virus .................................................68
Figure 8: Sequencing result confirms the target mutation ..............................69
Figure 9: Expression of hCBS, his CBS, D47E CBS and his D47E CBS in
Sf21 cells ..................................................................................................70
Figure 10: Overexpression of CBS protein analyzed in SDS-PAGE by
Comassie Blue ..........................................................................................71
Figure 11: Expression of CBS protein detected by Immunoblotting ..............72
Figure 12:Expression of D47ECBS protein detected by Immunoblotting ......73
Figure 13: Expression of hisCBS protein detected by Immunoblotting ..........74
Figure 14: Time course study to determine the infection and harvest condition
for abundant protein harvest ....................................................................76
Figure 15: Purification of CBS protein by Ni-NTA column ...........................77
Figure 16: Spectrophotometric analysis of human Cystathionine beta-synthase
..................................................................................................................78
Figure 17: Cross linking of wild and mutant CBS protein ..............................79
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