Establishment of recombinant protein production and cell-based assay system for research on MFGE8 protein = MFGE8 단백질 연구를 위한 재조합 단백질 생산 및 세포 기반 스크리닝 시스템 구축|RISS 상세보기

다국어 초록 (Multilingual Abstract)

The global pharmaceutical market is experiencing a surge in demand for biopharmaceuticals due to an aging population and an increase in chronic diseases. Protein-based drugs, targeting specific molecules, exhibit fewer side effects than traditional drugs. However, overcoming their high cost and short half-life poses significant research challenges. In this study, I investigated refined approaches to augment the production of Milk Fat Globule-EGF Factor 8 (MFG-E8) protein, prolong its in vivo half-life, and evaluate its anti-inflammatory effects. Additionally, I developed a cell-based assay system employing a lentiviral platform to screen for novel compounds influencing the expression of MFGE8 mRNA. Three structural designs, including wild-type MFG-E8, MSA-fused MFG-E8, and Fc-fused MFG-E8, were developed to assess protein expression levels through modifications of a critical element influencing production. My results revealed differences in expression levels due to variations in the expression vector. MSA-fused MFG-E8 exhibited significantly prolonged half-life compared to Wild-type MFG-E8. However, limitations in Fc-fused MFG-E8 indicated the need for further investigation into design elements and positioning in future studies. MFG-E8 administered intraperitoneally in mouse experiments demonstrated potential anti-inflammatory properties in brain tissues. Though uncertain regarding Blood-Brain Barrier (BBB) penetration, my findings suggest the possibility of indirect influence by these drugs. Furthermore, I developed a rapid cell-based assay system to screen compounds that increase MFGE8 expression and regulate inflammatory mediators, hinting at adjunct therapy potential. Optimizing the production system, extending the half-life, and exploring effective compounds indicate the potential of MFGE8 as a novel therapeutic in biopharmaceuticals.

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국문 초록 (Abstract)

글로벌 의약품 시장은 고령 인구 증가와 만성 질환 증가로 인해 바이오 의약품에 대한 수요가 급증하고 있다. 특정 분자를 표적으로 하는 단백질 기반 약물은 기존 약물에 비해 부작용이 적으나 높은 비용과 짧은 반감기를 극복하는 것은 중요한 연구 과제이다. 본 연구에서는 Milk Fat Globule-EGF Factor 8 (MFG-E8)의 재조합 단백질 생산을 늘리고 생체 내 반감기를 연장하는 전략을 적용하여 생산된 단백질을 평가하였다. 또한, MFGE8의 상위 조절 인자와 물질을 탐색하기 위한 렌티바이러스 기술을 적용한 세포 기반 스크리닝 시스템을 구축하여 검증하였다.
MFG-E8, MSA-융합 MFG-E8 그리고 Fc-융합 MFG-E8을 포함한 세 가지 구조적 설계를 개발하여 생산에 영향을 주는 중요 요소를 수정함으로써 단백질 발현의 수준을 평가했다. 이 결과는 발현 벡터의 차이로 인한 발현 수준의 차이로 검증되었다. MSA-융합 MFG-E8은 무변형MFG-E8에 비해 현저히 연장된 반감기를 보였다. 그러나 Fc-융합 MFG-8E의 한계로 인해 향후 연구에서 구조적 요소 대한 추가적인 조사와 기술적 적용의 필요성이 대두되었다. 마우스 실험에서 복강내 주입된MFG-E8은 뇌 조직에서 잠재적인 항염증 반응을 유도하였다. 혈액-뇌 장벽 투과에 대해서는 불확실하지만, 이러한 발견은 단백질에 의한 직, 간접적인 영향의 가능성을 시사한다. 구축된 세포 기반 MFGE8스크리닝 시스템을 활용하여 염증반응과 연관된 천연물 단일 물질을 성공적으로 스크리닝 선별하였다. 이러한 선행연구를 통하여 향후 MFGE8의 단백질 의약품으로서 유효성 평가와 MFGE8을 조절하는 새로운 물질을 탐색하는 연구에 활용할 수 있을 것으로 기대된다.

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글로벌 의약품 시장은 고령 인구 증가와 만성 질환 증가로 인해 바이오 의약품에 대한 수요가 급증하고 있다. 특정 분자를 표적으로 하는 단백질 기반 약물은 기존 약물에 비해 부작용이 적...

목차 (Table of Contents)

Abstract ⅰ
Table of contents ii
List of Tables v
List of Figures vi
1. Introduction 1

Abstract ⅰ
Table of contents ii
List of Tables v
List of Figures vi
1. Introduction 1
2. Materials and Method 9
2.1 Experimental animals and sample collection 9
2.2 Measurement of Protein Half-life 9
2.3 Cell Cultures 10
2.4 Differentiation and Preparation of RNA 10
2.5 Construction of Non-viral and Lentiviral Expression Plasmids 11
2.6 Transfection and Expression test 12
2.7 Large-Scale Transfection and Purification 13
2.8 Lentiviral production and Establishment of THP-1 Polyclone Regulating Luciferase Expression by MFG-E8 Promoter 14
2.9 qRT-PCR Analysis 15
2.10 Cell-Based Screening System 16
3. Results_Part-1.Production of MFGE8 recombinant protein with extended half life 17
3.1 Flow Chart for the Transient Mammalian Expression System 18
3.2 Construction of MFG-E8 Expression Plasmids 20
3.3 Replacement of the MFG-E8 Expression Plasmid with the pCEP4 29
3.4 Comparison of MFG-E8 Protein Expression in pLGNF and pCEP4 Plasmid 32
3.5 Large-Scale Transfection and Purification 35
3.6 Evaluation of Half-Life Extension in Mice 37
3.7 Anti-inflammatory Effect of MFG-E8 Protein in Mice brain 39
Part-2.Establishment of Cell-based assay system for research on MFGE8 protein
3.8 Reporter Cell line Development Workflow 44
3.9 Construction of hMFG-E8 Promoter Containing Lenti-Vector 46
3.10 Flow Chart for the Lentiviral Particle and Stable Cell Generation System 51
3.11 PMA-Induced Differentiation of THP-1 Monocyte into Macrophages 53
3.12 Effect of Dexamethasone on Transcriptional Activity of MFG-E8 Promoter in THP-1 Cells 54
3.13 Effect of Single Compounds on Transcriptional Activity of MFG-E8 Promoter 56
3.14 Single Compounds Treatment Induces an Upregulation of MFG-E8 Expression in THP-1 Cells 59
3.15 Inhibition of Inflammatory Mediator Expression in THP-1 Cells through Single Compound Treatment 62
4. Discussion 64
5. Reference 68
국문초록 73

참고문헌 (Reference)

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