Purification and Characterization of a Non-Specific Lipid Transfer Protein 1 (nsLTP1) from Local Coffee Beans (Coffea arabica)

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研究生：	林惠雯 Lim, Hwee Woon
論文名稱：	Purification and Characterization of a Non-Specific Lipid Transfer Protein 1 (nsLTP1) from Local Coffee Beans (Coffea arabica) 咖啡豆非專一性脂質運輸蛋白之純化與功能特性分析
指導教授：	呂平江 Lyu, Ping-Chiang
口試委員:	林彩雲 Lin, Tsai-Yun 張家靖 Chang, Chia-Ching
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	72
中文關鍵詞：	非專一性脂質運輸蛋白、咖啡豆
外文關鍵詞：	non-specific lipid transfer protein 1, coffee beans
相關次數：	點閱：3 下載：0
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植物非專一性脂質運輸蛋白1(non-specific lipid transfer proteins 1, nsLTP1)是一種小分子的鹼性蛋白質。試管內實驗證明nsLTP1具有促進脂質轉運和交換的能力，所以推測其與角質的形成有關。根據先前的研究指出巴西Coffea canephora豆中的CcLTP1具有抑制哺乳類α-澱粉酶的功能以及抗真菌的特性。然而，CcLTP1的生物化學和生物物理特性還尚未清楚。所以我們研究目的是純化出台灣本土Coffea arabica咖啡豆中的nsLTP1，並闡明nsLTP1的特性以及結構和功能之間潛在的關係。在這篇研究中，我們成功純化出兩個分子量約為9.3 kDa的protein isoforms，分別命名為nsLTP1A和nsLTP1B，並利用N端定序確定nsLTP1A屬於nsLTP protein family。根據圓二色譜分析，nsLTP1A和nsLTP1B具有高穩定性的α-螺旋二級結構，並在抗菌實驗中發現nsLTP1A和nsLTP1B的混和樣品具有抑制熱帶假絲酵母(Candida tropicalis)和白色念珠菌(Candida albicans)的作用。另外，在脂質轉移的螢光實驗中發現nsLTP1A和nsLTP1B有不同的脂質轉移能力。不同於巴西Coffea canephora豆研究的是nsLTP1A和nsLTP1B均不具有抑制哺乳動物的α-澱粉酶活性。最後，本研究已成功鑑定出nsLTP1的cDNA序列。

Plant non-specific lipid transfer proteins 1 (nsLTP1) are small and basic proteins. In vitro studies showed that nsLTP1 facilitates lipid binding and transfer, so it is presumably related to the formation of cutin. According to the previous other study, a novel nsLTP, named CcLTP1 purified from Brazil Coffea canephora seeds, exhibited the unique mammalian α-amylase inhibitory and antifungal properties. However, the biochemical and biophysical characterization of CcLTP1 are still unclear. The aims of our research are to characterize nsLTP1 from Taiwan local Coffea arabica and to elucidate the potential relationship between the biological functions and the protein structure. Two isoforms of nsLTP1 with the molecular mass of 9.3 kDa, named nsLTP1A and nsLTP1B were purified from local Coffea arabica. The N-terminal 18-amino acids of nsLTP1A, ITCQQVTHELEPCVPYLT, were identified and exhibited a unique nsLTP pattern. Circular dichroism analysis suggested both isoforms have α-helical conformation with high protein stability. The mixture of nsLTP1A and nsLTP1B exhibited antifungal effect toward Candida tropicalis and Candida albicans, however, the lipid transfer abilities of both isoforms were different. Unlike the study of Brazil Coffea canephora, both isoforms possessed no inhibitory activity against mammalian α-amylase activity. Finally, we have successfully purified, identified and evaluated the characteristics of Coffea arabica nsLTP1A and nsLTP1B in this study. cDNA sequence of local Coffea arabica nsLTP1 has also been identified.

Table of Contents………………………………………………………………………….    i
Abstract……………………………………………………………………………………    iv
摘要………………………………………………………………………………………..    v
Abbreviations.......................................................................................................................    vi
List of Tables………………………………………………………………………………    vii
List of Figures……………………………………………………………………………..    viii
Chapter 1: Introduction………………………………………………………………….    1
1    Plant non-specific lipid transfer proteins (nsLTPs)……………………………….    1
2    Functions of nsLTP………………………………………………………………..    2
2.1    Lipid binding and transfer ability………………………………………….    2
2.2    Biosynthesis of cutin……………………………………………………….    2
2.3    Plant defense against biotic and abiotic stress…………………………….    2
2.4    Beer brewing……………………………………………………………….    3
2.5    Fruit allergens………………………………………………………………    3
3    Coffee bean…………………………………………………………………………    4
4    Objective of the study    ……………………………………………………………...    4
Chapter 2: Materials and Methods………………………………………………………    5
1    Purification of nsLTP from Coffea arabica………………………………………..    5
2    Identification of nsLTP1A and nsLTP1B………………………………………….    6
2.1    Tricine SDS-PAGE and MALDI-TOF……………………………………..    6
2.2    N-terminal amino acid sequencing…………………………………………    6
2.3    Quantification of protein concentration……………………………………    7
3    Biophysical properties of nsLTP1A and nsLTP1B………………………………..    7
3.1    Circular dichroism (CD)……………………………………………………    7
3.2     Intrinsic fluorescence………………………………………………………    8
3.3     Extrinsic fluorescence (ANS binding)……………………………………    8
4     Functional assays…………………………………………………………………    9
4.1     Lipid binding assay……………………………………………………….    9
4.2     Lipid transfer assay……………………………………………………….    10
4.3    Human salivary α-amylase inhibition assay……………………………...    10
4.4     Antimicrobial assay……………………………………………………….    11
5    Total RNA extraction……………………………………………………………..    12
6    Reverse transcription of mRNA to cDNA………………………………………...    14
7    PCR the nsLTP1 gene sequence…………………………………………………...    14
8    Construction of homology modeling………………………………………………    15
9     Molecular docking…………………………………………………………………    15
Chapter 3: Results and Discussions……………………………………………………...    16
1     Purification and identification of nsLTP1A and nsLTP1B………………………..    16
2     Secondary structure of nsLTP1A and nsLTP1B…………………………………..    17
3    Stability of nsLTP1A and nsLTP1B……………………………………………….    18
3.1     Thermal denaturation………………………………………………………    18
3.2    pH titration…………………………………………………………………    18
3.3     Chemical denaturation……………………………………………………..    18
4     Hydrophobicity of nsLTP1A and nsLTP1B……………………………………….    19
5     Functional studies of nsLTP1A and nsLTP1B…………………………………….    20
5.1     Lipid binding ability………………………………………………………..    20
5.2     Lipid transfer ability……………………………………………………….    21
5.3     Human salivary α-amylase inhibition    ……………………………………..    22
5.4     Antimicrobial activity    ……………………………………………………...    22
6    Validation of the nsLTP1 gene sequence…………………………………………..    23
7     Molecular modeling and docking…………………………………………………..    25
Chapter 4: Conclusions…………………………………………………………………...    26
References…………………………………………………………………………………    28
Attached Tables……………………………………………………………………………    32
Attached Figures…………………………………………………………………………..    37

                                

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