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研究生:楊致誠
研究生(外文):Chih-Cheng Yang
論文名稱:臺灣眼鏡蛇毒中心臟毒蛋白II的蛋白質表現,定點突變與結構,功能研究
論文名稱(外文):Study of Cardiotoxin II from Taiwan Cobra : Protein Expression, Site-directed Mutagenesis, Structure and Function
指導教授:邱式鴻邱式鴻引用關係
指導教授(外文):Shyh-Horng Chiou
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
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:2000
畢業學年度:88
語文別:英文
論文頁數:100
中文關鍵詞:心臟毒蛋白II心臟毒蛋白蛋白質表現定點突變
外文關鍵詞:cardiotoxin IICTXprotein expressionSite-directed Mutagenesis
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我們利用以PCR技術合成出的mdctxII基因,其中包含了能夠轉譯出心臟毒蛋白II的核酸密碼,以及一段可以在表現毒素的氮端(N-terminus)轉譯出(Asp)4-Lys的核酸序列。這段基因轉殖到pQE-30載體上後,再將載體送入大腸桿菌(Escherichia coli)中以表現所要探討的心臟毒蛋白II(CTX II)。轉殖後篩選出含有心臟毒蛋白II基因的菌體,再利用核酸定序儀確定這段基因的序列是正確的。我們利用此菌株Y1在大量養殖的過程中,以IPTG誘導氮端含有組胺酸標示物(His-tag)的心臟毒蛋白II的表現。在接下來的純化過程中,首先用親和層析管柱(affinity chromatography)初步分離,然後在特定的條件下進行重新折疊(refolding)並進一步以高效能層析法(HPLC)純化,在此經由質譜儀(mass spectrometry)證實其分子量是正確的,最後再通過分子篩管柱(size-exclusion chromatography),所純化的蛋白利用圓偏光二色光譜(circular dichroism)和溶血活性測試,其結果與從粗毒純化得到的心臟毒蛋白II大致相同。
從電腦模擬的圖像中可以發現位於第35個胺基酸的離胺酸(lysine)在分子表面上形成一正電區域,為了瞭解這區域對心臟毒蛋白活性的影響,我們利用定點突變(site-directed mutagenesis)的技術得到兩個突變株,其中一株菌所表現的蛋白質(K35W),是將第35個胺基酸由離胺酸(lysine)變成色胺酸(tryptophan),以期改變這區域的疏水性質;另一株表現的蛋白(K35L)則是離胺酸變成白胺酸(leucine),將其帶正電性質改成不帶電。在實驗中,第35個胺基酸改變成色胺酸後造成無法重新折疊而沈澱,若改變成白胺酸後,圓偏光二色光譜證明其重新折疊是正確的,但是毒蛋白的溶血活性盡失,然而紅血球會聚集(aggregation )且黏附在試管內壁,有可能是突變造成心臟毒蛋白II使血球破裂的功能喪失而仍維持有與血球結合的能力。因此我們推論第35胺基酸可能是引起細胞破裂的胺基酸之一,當其正電性消去後造成溶血能力的喪失,而若性質改變太劇烈,亦會造成無法重新折疊的情況,所以第35個胺基酸對於心臟毒蛋白II的結構及功能有重要的影響。

The mdctxII gene, which encodes cardiotoxin II (CTX II) and an N-terminal extension of (Asp)4-Lys and His-tag fragments located at the 5′-end noncoding region, was constructed using an artificial synthetic approach. The mdctxII gene generated by means of polymerase chain reaction (PCR) was ligated into pQE30 expression vector and then transformed the vector into E. coli. A positive clone, named Y1, which contains the exact nucleotide sequence as CTX II, was obtained after multiple screening steps and used for the expression of mCTX II. The expressed protein was first purified by affinity chromatography, followed by refolding under defined conditions and further purification of the refolded product on RP-HPLC. The expressed product was then confirmed according to its molecular weight by mass spectrometry. In order to increase resolution, the lyophilized peak fraction of RP-HPLC was redissolved in 100 mM NaCl and applied to a gel filtration column. Finally, the fractions collected from gel filtration column were confirmed to have a similar molecular structure by circular dichroism to native CTX II isolated and purified from crude venom. The activity of expressed mCTX II was also found to possess high hemolytic activity against rabbit reticulocytes similar to natural CTX II, corroborating that expressed mCTX II has refolded correctly into active conformation.
In order to identify amino acids specific for CTX II hemolytic activity, the role of a conserved basic residue in hemolytic activity was tested. Using the molecular modeling program, Delphi, the basic residue Lys 35 was shown to provide an area of positive charge on the surface of toxin molecule. In order to study the role of this residue in the biological activity, site-directed mutants were prepared and hemolytic activity was measured. By replacing Lys 35 to construct two mutants, K35W and K35L, resulted in the change of hydrophobicity and loss of positive charge on CTX II, respectively. Mutation of Lys35 to Trp destabilized the toxin and caused aggregation during a refolding step. Although mutation of Lys35 to Leu was shown to refold correctly as judged by CD spectra, the hemolytic activity was almost lost. However, the red blood cells treated with K35L CTX II mutant became aggregated and adhered to the surface of tubes. Therefore, the mutant CTX maintains its binding capacity even though it has lost hemolytic activity. It is proposed that Lys35 on the convex side may be one of the residues involved in the rupture of red blood cells. Decreasing the positive surface potential provided by Lys35 resulted in the loss of hemolytic activity and increasing hydrophobicity of Lys35 resulted in toxin aggregation during the refolding process. Therefore, it is believed that Lys 35 plays an important role in the function and activity of CTX II.

中文摘要…………………………………………………………… 1
Abstract ………………………………………………………… 2
List of Abbreviations ……………………………………….. 4
Chapter 1 : Introduction…………………………………….. 5
Motivations of this thesis………………………….. 5
Minireview of snake venoms…………………………… 6
Isolation and purification of cardiotoxin………… 7
Structure of cardiotoxins……………………………… 7
Possible mechanisms of cell lysis……………………12
Binding and penetration model………………………12
Membrane permeability model…………………….. 14
Pore-formation model……………………………. 14
Cardiotoxin-phospholipase synergism model………14
Basic residues in CTXs………………………………… 15
Figures………………………………………………………17
Chapter 2 : The Purification of Venom Toxins……………21
Materials and Methods ………………………………… 21
Cation-exchange chromatography…………………… 21
Reverse-phase HPLC…………………………………….22
Molecular weight analysis by Mass…………………22
Circular dichroism measurement…………………… 22
Results and Discussion………………………………… 23
Figures………………………………………………………26
Chapter 3 : The Expression and
Purification of Cardiotoxin II… 33
Materials and Methods ………………………………… 33
Bacterial strains, plasmids, and chemicals…… 33
Preparation of competent M15[pREP4] cells………34
Transformation………………………………………… 35
Screen for positive clones………………………… 35
Sequence analysis………………………………………35
Small-scale expression of cardiotoxin II……… 35
SDS-polyacrylamide gel electrophoresis………… 36
Growing large-scale expression culture………… 37
Denaturing purification of insoluble proteins…37
Affinity purification on Ni chelate column…… 37
Refolding…………………………………………………38
High pressure liquid chromatography………………38
Gel-filtration chromatography …………………… 38
Results and Discussion………………………………… 39
Figures………………………………………………………42
Chapter 4 : The Molecular Modeling of Cardiotoxin II
and its Mutant……………………………………. 52
Materials and Methods ………………………………… 53
Results and Discussion………………………………… 53
Figures………………………………………………………55
Chapter 5 : Site-directed
Mutagenesis of Cardiotoxin II… 60
Materials and Methods ………………………………… 60
Site-directed mutagenesis……………………………60
Elution and restriction enzyme digestion……… 61
Ligation of the mutated gene into pQE-30 vector…61
Transformation (for cloning and plasmid selection)…... 61
Miniprep………………………………………………… 62
Immunoblotting………………………………………… 62
Results and Discussion………………………………… 63
Figures………………………………………………………66
Chapter 6 : Biological Activity of Cardiotoxin II…… 76
Materials and Methods ………………………………… 76
Haemolytic activity……………………………………76
Results and Discussion………………………………… 76
Figures………………………………………………………78
Chapter 7 : The Cleavage of His-tag in mCTX II…………81
Materials and Methods ………………………………… 81
Cleavage by enterokinase…………………………… 81
Electroblotting of protein bands from native
polyacrylamide gels onto PVDF membranes…… 81
Protein sequence analysis ………………………… 82
Results and Discussion………………………………… 82
Figures………………………………………………………84
Chapter 8: Conclusions and Prospect……………………… 89
References………………………………………………………… 91

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