建立TurboID鄰近標記方法以鑑定根癌農桿菌TssC40的交互作用蛋白質

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研究生：	鄭勻媜 Yun-Chen Cheng
論文名稱：	建立TurboID鄰近標記方法以鑑定根癌農桿菌TssC40的交互作用蛋白質 Establishment of TurboID Proximity Labeling to identify TssC40-interacting proteins in Agrobacterium tumefaciens
指導教授：	賴爾珉 Erh-Min Lai 吳少傑 Shaw-Jye Wu
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
論文出版年：	2022
畢業學年度：	111
語文別：	英文
論文頁數：	115
中文關鍵詞：	根癌農桿菌、第六型分泌系統、交互作用蛋白質、鄰近標記方法
外文關鍵詞：	TurboID, Proximity Labeling
相關次數：	點閱：12 下載：0
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第六型分泌系統(type VI secretion system, T6SS)存在於約25%的革蘭氏陰性菌中，是一種對抗細菌的武器。第六型分泌系統所分泌的效應子或毒素蛋白質可以從攻擊細胞的細胞質通過細胞膜遞送到所接觸的目標細胞中。第六型分泌系統由至少13個核心蛋白質所組成，稱為TssA-TssM (type VI secretion A-M)，它們的構造分別為膜蛋白複合體、底座蛋白、外鞘和攜帶效應器的注射裝置，組成一個可收縮的分子武器。根癌農桿菌是植物的病原菌，會造成冠癭病，也是基因轉移的工具。第六型分泌系統普遍存在許多根癌農桿菌基因複合種的基因體中，除了包含具高度保守性的Tss核心蛋白質外，根癌農桿菌的第六型分泌系統也擁有一些獨特的核心蛋白質，但現今對其分子功能的了解有限。在這項研究中，我的目標是使用一種稱為TurboID的鄰近標記方法，利用其生物素化酶來標記鄰近蛋白質，以鑑定三個第六型分泌系統核心蛋白質，分別為底座蛋白質TssA和TssK以及根癌農桿菌C58的獨特核心蛋白TssC40，與其他第六型分泌系統蛋白質之間的交互作用。我首先將TurboID分別與TssA、TssK和TssC40融合，回補到相對應的突變體中，並以西方墨點法確認其表現第六型分泌系統是否恢復分泌能力。結果表明，TssC40-HA-Turbo可以完全回補突變株的第六型分泌能力，而TurboID融合的TssA和TssK則不能回補。因此，針對TssC40-HA-Turbo進行Streptavidin下拉生物素化蛋白質，再以質譜法鑑定蛋白質身份。我發現，與僅表達TurboID或TssC40的突變體菌株相比，表達TssC40-HA-Turbo的突變體菌株其TssA、TssB、TssC41在Streptavidin下拉的樣品中有較高的量，顯示TssC40-HA-Turbo與TssA、TssB、TssC41蛋白質可能有交互作用。最後，再以免疫共沉澱和蛋白質下拉實驗驗證TssC40分別與TssA、TssB和TssC41有交互作用。

Type Ⅵ secretion system (T6SS) is an antibacterial weapon found in ~25% of Gram-negative bacteria. T6SS effectors or toxin proteins can be delivered from the cytoplasm of the attacker cell through the cellular envelope into the target cell in a contact-dependent manner. T6SS is composed of at least 13 conserved core proteins, named as TssA-TssM (type VI secretion A-M), which are assembled into a contractile molecular weapon consisting of a membrane complex, baseplate, sheath and puncturing device carrying effectors. T6SS is highly conserved in the Agrobacterium tumefaciens genomospecies complex, a causal agent of crown gall disease and gene transfer tool. While most Tss core proteins encoded in A. tumefaciens are highly conserved among T6SS-containing bacteria, A. tumefaciens harbors some unique core proteins with limited knowledge about their molecular functions. In this study, I aim to use a proximity labeling method termed TurboID, a promiscuous biotinylating enzyme, to identify the interaction proteins of three T6SS core proteins, TssA and TssK baseplate components, and a unique core protein TssC40 of A. tumefaciens C58. I first generated TurboID fusion to TssA, TssK, and TssC40 and determine their expression and functionality by western blotting of cellular and extracellular fractions in each of the respective mutants. The results showed that TssC40-HA-Turbo could fully rescue the T6SS secretion in the mutant while TssA and TssK fused with TurboID could not. Thus, TssC40-HA-Turbo expressing strain was subjected for protein extraction followed by biotinylating and Streptavidin pulldown to identify biotinylated proteins by mass spectrometry. I found that TssA, TssB, TssC41 were identified as enriched proteins in the TssC40-HA-Turbo expression strain as compared to strains expression TurboID only or TssC without fusion to TurboID. The interactions of TssC40 with TssA, TssB, and TssC41 were validated by co-immunoprecipitation and co-purification experiments.

中文摘要    I
Abstract    III
致謝    V
List of figures XII
List of tables XIV
Introduction    1
1. Bacterial secretion systems    1
1.1 Type VI secretion system (T6SS)    1
2. Agrobacterium tumefaciens    3
3. T6SS in A. tumefaciens    4
3.1 TssA and TssK baseplate components, and a unique core protein TssC40 of A. tumefaciens C58    4
4. Proximity labeling    6
4.1    Methods of proximity labeling    6
4.2    Optimization of biotin-based proximity labeling    7
Materials and methods    9
1. Bacterial strains and growth conditions    9
2. Cloning techniques and plasmid construction    9
3. Type VI secretion assay    10
4. Streptavidin pull-down assay    11
5. Identification of biotinylated proteins    13
5.1 Sample preparation    13
5.2 Mass Spectrometry Method    13
5.3 Data analysis    14
6. Co-immunoprecipitation (IP) assay    15
7. Co-purification in E. coli    17
8. GST pull-down assay    18
9. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis    19
10. Sequence analysis    20
Results    22
1. Generation of TurboID vector    22
2. Protein expression analysis of pRL662-HA-Turbo and pRL662-sfGFP-HA-Turbo in A. tumefaciens    22
3. Generation and functional analysis of constructs expressing TurboID fused to TssA, TssK, and TssC40    23
4. Biotin concentration optimized for Turbo-ID experiment    25
5. Specific biotinyltion and streptavidin pull-down of TssC40-HA-Turbo and HA-Turbo    25
6. Mass spectrometry identification of streptavidin pull-down proteins    26
7. TssA, TssB and TssC41 were identified as enriched T6SS proteins in the TssC40-HA-Turbo expression strain    27
8. Sequence analysis and domain prediction of TssC40    27
9. Validation of TurboID results of TssC40 interacting with TssA, TssB and TssC41            28
Discussion    31
References 34
Appendices    27
1. Medium and buffer used in this study    27
1.1 Lysogeny broth (LB)    27
1.2 523    27
1.3 AB-MES    27
1.4 TE buffer    28
1.5 Sample loading buffer (SSB)    28
1.6 RIPA Lysis buffer    28
1.7 Lysis buffer (Co-purification in E. coli)    28
1.8 Washing buffer (Co-purification in E. coli)    29
1.9 Elution buffer    29
1.10 Lysis buffer (purification of GST-tagged proteins)    29
1.11 Washing buffer (GST pull-down)    29
1.12 1X Transfer buffer 30
1.13 TBST    30
Appendix Figure 1. The plasmid maps of pRL662-sfGFP-HA-Turbo and pRL662-HA-Turbo    31
Appendix Figure 2. The plasmid maps of of TssA, TssC40, TssK fused to HA-Turbo or sfGFP-HA-Turbo on pRL662 shown in Appendix Figure 1. Each gene contains its own ribosome binding site.    33
Appendix Figure 3. Repeat data of Co-IP of TssA and TssB withTssC40-HA-Turbo by anti-HA using Wash buffer B with 3 wash times.    34
3. Appendix Tables    1
3.1 All the results of comparison with the protein abundance by proteomic core laboratory……………………………………………………………………………………..1
3.2 All the results of the comparison using PRM with the T6SS of A. tumefaciens C58 protein abundance by proteomic core laboratory.    9
4. Abbreviation in this study    1


List of figures
Figure 1. T6SS gene cluster and illustration of T6SS apparatus of A . tumefaciens C58.    1
Figure 2. Construct design for TurboID and its fusion to TssA, TssK, and TssC40    3
Figure 3. The workflow of TurboID for identification of biotinylated proteins    5
Figure 4. The workflow of Co-Immunoprecipitation by anti-HA    6
Figure 5. The workflow of E. coli co-purification    7
Figure 6. GST pull-down    8
Figure 7. Expression of HA-Turbo and sfGFP-HA-Turbo in A. tumefaciens    9
Figure 8. The expression and secretion of TssA fusion proteins in tssA    11
Figure 9. The expression and secretion of TssK fusion proteins in tssK    12
Figure 10. The expression and secretion of TssC40 fusion proteins in tssC40    13
Figure 11. Biotin concentration optimized for Turbo-ID experiment    14
Figure 12. TssC40-HA-Turbo expression strain can be detected by streptavidin pulldown        15
Figure 13. Domain prediction and sequence alignment of TssC40    17
Figure 14. Protein prediction of TssC40 in A. tumefaciens C58    20
Figure 15. Co-IP of TssA and TssB withTssC40-HA-Turbo by anti-HA using Wash buffer A        21
Figure 16. Co-IP of TssA and TssB withTssC40-HA-Turbo by anti-HA using Wash buffer B        22
Figure 17. Co-IP of TssA and TssB withTssC40-HA-Turbo by anti-HA using Wash buffer C        23
Figure 18. GST pull-down for interaction analysis between TssC40 with TssB or TssA.    24
Figure 19. E. coli co-expression of TssC40 and TssB.    26

List of Tables
Table 1. Bacterial strains    38 
Table 2. Plasmid information    39
Table 3. Primer information    43
Table 4. The recipe and condition of Gibson assembly DNA polymerase    45
Table 5. The recipe and condition for PCR with Taq DNA polymerase    46
Table 6. The recipe and condition for PCR with KAPA DNA polymerase    47
Table 7. The comparison with the protein abundance by proteomic core laboratory.    1
Table 8. The comparison using PRM with the T6SS of A. tumefaciens C58 protein abundance by proteomic core laboratory.    5

                                

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