Analysis of THSD7A functional domain expressed by baculoviral system

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研究生：	吳曉鈞 Wu, Hsiao-Chun
論文名稱：	Analysis of THSD7A functional domain expressed by baculoviral system 分析以昆蟲病毒系統表達之凝血酶敏感蛋白區域包含7A的功能性片段
指導教授：	莊永仁 Chuang, Yung-Jen
口試委員:
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：	2010
畢業學年度：	98
語文別：	英文
論文頁數：	56
中文關鍵詞：	凝血酶敏感蛋白區域包含 7A 、桿狀病毒-昆蟲細胞表現系統、人類臍帶靜脈內皮細胞、細胞遷移
外文關鍵詞：	THSD7A, baculovirus-insect cell expression system, HUVEC, cell migration
相關次數：	點閱：3 下載：0
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內皮細胞的方向性遷移對血管及其形態的發育扮演重要的角色。它受到許多引導因子嚴密的調控。先前，我們發現一個新穎蛋白: 凝血酶敏感蛋白區域包含 7A (Thrombospondin Type-1 Domain Containing 7A; THSD7A) 可能參與上述過程，但是其結構與功能的關聯機制尚未被深入了解。利用生物資訊分析的結果顯示，THSD7A被預測為一個包含十一個凝血酶敏感蛋白重複區域 (TSR) 以及一個RGD序列的穿膜醣蛋白。這些特徵暗示THSD7A可能參與細胞的移動或細胞與胞外基質的交互作用。在我們先前的研究中指出，當斑馬魚體內THSD7A的表現減少時，會導致體節間血管 (ISV) 不正常的分歧與生長延遲。另一方面，我們觀察到在血管前端的內皮尖端細胞 (endothelial tip cell) 上生成過多的絲狀偽足 (filopodia)，其形成被視為調控遷移方向的關鍵因素。同時，我們也發現THSD7A的轉錄產物會表現在斑馬魚的神經系統中。基於上述的發現，我們假設THSD7A可能是一個從神經組織分泌出的血管引導蛋白，並且會在血管發育過程中扮演外源性負向調控內皮尖端細胞方向性遷移的因子。本篇論文研究的主要目的是利用活體外的分析方式找出THSD7A的功能性片段。首先，為了釐清人類THSD7A的轉譯後修飾與其同源異構體 (isoform)，我們藉由哺乳類細胞株表現其蛋白。我們亦利用桿狀病毒-昆蟲細胞表現系統成功建構THSD7A截切片段TF2之載體並表現蛋白。最後，我們利用穿透式細胞遷移試驗 (transwell migration assay) 與管柱生成試驗 (tube-like formation assay) 兩種與血管發育相關的活體外實驗，測試TF2的功能。結果顯示，THSD7A是一個會被分泌到細胞外的醣蛋白。TF2-His融合蛋白可以成功地藉由培養在Express 5培養基的High5 昆蟲細胞表現並以適當的條件純化之。然而，我們發現 TF2不會調控人類臍帶靜脈內皮細胞的遷移與管柱生成能力。這個結果暗示我們THSD7A的功能性片段可能不是座落在TF2上。

Directed migration of endothelial cell (EC) plays a critical role in vascular growth and patterning processes. It is tightly regulated by various guidance cues and molecules. Previously, we identified a novel protein, Thrombospondin Type-1 Domain Containing 7A (THSD7A), which may involved in this process, but not much is known regarding its structural-functional mechanism. By bioinformatic analysis, THSD7A was predicted as a membrane glycoprotein which contains eleven thrombospondin-type-1 repeats (TSR) and one RGD motif. These characters imply THSD7A may be involved in cell migration and cell-to-ECM interactions. In our previous study, we found out that the down-regulation of zTHSD7A (THSD7A homolog in zebrafish) gene expression resulted in abnormal branching and stalled of intersegmental vessel (ISV). On the other hand, the endothelial tip cell at the leading front of growing vessel showed excessive filopodia formation which was thought to guide its direction. We also discovered that Thsd7a transcript was expressed in zebrafish nervous system. Based on these findings, we hypothesize that THSD7A may be a vessel guidance protein secreted by neural tissue and act as an exogenous negative regulator of the directed migration of endothelial tip cell during vascular growth. The specific aim of this thesis study is focusing on analyzing the functional domain of THSD7A in vitro. First, recombinant full-length human THSD7A was expressed by mammalian cells to reveal the isoforms of THSD7A and modification. Baculoviral vectors containing truncated fragment 2 (TF2) of THSD7A was then constructed and used to express the target protein region by insect cells system. The resulting TF2 were then tested with two in vitro angiogenic assays, which are the transwell migration assay and tube-like formation assay. These results demonstrated that there was a secreted form of THSD7A which was glycosylated. His-tagged TF2 (TF2-His) can indeed be expressed by High5 insect cells in Express 5 medium successfully, and an optimal purification procedure was established. However, we found TF2 has no activity on regulating the migration and tube-formation of HUVEC. This suggests that the functional domain of THSD7A may not locate within the TF2 region.

摘要    I
Abstract    II
List of contents    III
List of figures    V
List of supplemental figures    VI
1. Introduction    1
1.1 Angiogenesis and its regulatory factors    1
1.2 The process of angiogenesis and endothelial cell directed migration    1
1.3 Thrombospondin structural homology repeat (TSR) and TSR-containing proteins    2
1.4 Thrombospondin Type 1 Domain Containing 7A (THSD7A) and its potential role in modulation of endothelial cells directed migration    2
1.5 N-glycosylation and its function    4
1.6 Baculovirus-insect cell expression system    4
1.7 The objective of this study    6
2.    Materials and Methods    7
2.1 Construction of protein expression vectors    7
2.2 Cell cultures of HEK293T, Sf9, High5 and HUVEC    7
2.3 Transfection of mammalian cells    8
2.4 Mammalian cell lysis and secretory protein collection    8
2.5 Cotransfection of insect cells with virus DNA and transfer plasmid DNA    9
2.6 Plaque assay    9
2.7 PCR analysis of recombinant virus DNA    11
2.8 Virus production and scale up    12
2.9 End-point dilution    12
2.10 Time course of recombinant protein expression    12
2.11 Western Blotting    13
2.12 Purification of TF2 recombinant protein    14
2.13 Sliver staining    15
2.14 HUVEC isolation    15
2.15 Functional assay of TF2 recombinant protein    16
3.    Result s    17
3.1 Analysis of the putative human THSD7A    17
3.2 Human THSD7A is a secreted protein    17
3.3 Human THSD7A is N-glycosylated in mammalian cells    18
3.4 Generation of TF2 recombinant virus    19
3.5 Expression and identification of TF2 recombinant protein in High5 cells    21
3.6 TF2 recombinant protein has an N-glycosylated form in High5 cells    23
3.7 Purification of TF2 recombinant protein by nickel affinity chromatography    24
3.8 Validation of the purified TF2 recombinant protein    25
3.9 TF2 recombinant protein does not affect HUVEC migration and tube formation    26
4.    Discussion    27
5.    References    31

List of figures
Figure 1: The predicted Thrombospondin Type1 Domain Containing 7A    36
Figure 2: Human THSD7A is an N-glycoprotein, and it can be a secreted protein.    37
Figure 3: The construction of truncated human THSD7A expression vector    38
Figure 4: The morphology of 4-7 days post-transfected Sf9 insect cells    39
Figure 5: Verify the recombinant virus DNA by polymerase chain reaction    40
Figure 6: The optimal expression time point and MOI of TF2-His can be recognized    41
Figure 7: TF2-His expressed by insect cells have an N-glycosylated form    42
Figure 8: The establishment of an optimized purification protocol with stepwise elution of TF2-His recombinant protein.    43
Figure 9: Identification of the TF2 recombinant protein    44
Figure 10: TF2 does not affect migration ability of endothelial cells    45
Figure 11: TF2 does not affect tube-like structure formation of endothelial cells    46

List of supplemental figures
Supplement 1: THSD7A may act as a repulsive regulator during ISV angiogenesis in vivo.    47
Supplement 2: Overview the process of recombinant protein production    51
Supplement 3: Blue plaque formation reveals the potential of Sf9 cells infected by TF2 recombinant virus    52
Supplement 4: Excessive MOI resulted in similar expression level of two forms of TF2 and have poor secretion ability    53
Supplement 5: The impurities of TF2 elution is contaminated by the damaged cells.    54
Supplement 6: The structure of TF2 may be destructed after truncation    55
Supplement 7: The predicted binding domain contain a TSR and a CD36-binding mitif    56

                                

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