Alboostumin: An Engineered Albumin as a Modular Scaffold for Drug Delivery
- Abstract
- Promising therapeutic proteins often fail to advance in the clinical evaluation due to poor half-life in the circulation. Thus, it is of great value to develop a drug carrier that can improve in vivo half-life of a payload without compromising its therapeutic activity. Serum proteins like immunoglobulin G (IgG) and albumin have been employed as long-acting drug carriers to exploit their innate ability to survive sustainably in the blood. Several Fc or albumin fusion proteins have demonstrated their potential in clinical trials. However, some proteins have complex folding pathways or conformation incompatible with genetic fusion to such carriers due to poor manufacturability or inefficaciousness. To provide a solution for this problem, this research was conducted by engineering human serum albumin (HSA) into a modular drug carrier by substituting its second domain with a self-assembling coil domain. The result indicated that the engineered HSA (eHSA) could self assemble with a payload protein modified by a complementary partner coil. Isothermal titration calorimetry (ITC), Size Exclusion Chromatography (SEC), and NATIVE-PAGE techniques have revealed the eHSA could tightly bind the payload with high affinity. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has an apoptotic effect on cancerous cells, and when it gets trimerized, its apoptotic activity would be upgraded. Cloning of trimerized TRAIL to the complementary coil as a more functional therapeutic compound and investigation by NATIVE-PAGE proved this big therapeutic molecule can still bind to eHSA with good affinity. Its interaction with FcRn by Microscale thermophoresis (MST) verify the eHSA would have a long half-life in the bloodstream.
전도유망한 치료 단백질은 종종 혈액 순환의 낮은 반감기로 인해 임상 평가에서 발전하지 못한다. 따라서, 치료 효과를 저해하지 않고 페이로드의 생체 내 반감기를 개선할 수 있는 약물 운반체를 개발하는 것이 매우 중요하다. 면역글로빈 G (IgG)와 알부민과 같은 혈청 단백질은 혈액에서 지속 가능한선천적능력으로 장기 작용을 위한 약물 운반체로 사용되어 왔다. 여러 Fc 또는 알부민 융접 단백질은 임상 시험에서 그 가능성을 입증했지만, 일부 단백질은 낮은 제조 가능성 또는 비효율성으로 인하여 그러한 운반체에 대해 유전적 융합과 양립할 수 없는 복잡한 접힘 경로 또는 형질을 가지고 있다. 이 문제에 대한 해결책을 제공하기 위해 본 연구는 인간 혈청 알부민(HSA)을 모듈식 약물 운반체로 엔지니어링하여 두 번째 도메인을 자가 조립 코일 도메인으로 대체함으로써 수행되었다. 결과는 엔지니어링된 HSA(eHSA)가 보완 파트너 코일에 수정된 치료 단백질과 자체 조립할 수 있음을 보여주었다. 등온 적정 열량계 (ITC), 크기 배제 크로마토그래피 (SEC) 및 Native-PAGE 기술은 eHSA가 높은 친화력으로 페이로드를 단단히 결합할 수 있음을 보여준다. 종양 괴사 인자와 관련된 세포사멸 유도 리간드(TRAIL)는 암세포에 세포사멸 효과가 있고, 그것이 삼합체를 형성하게 되면, 세포사멸 활성도가 증가될 것이다. 보다 기능적인 치료 화합물로서 TRAIL을 보완 코일에 융합하고 Native-PAGE에 의한 조사를 통해 이 큰 치료 분자가 양호한 친화력으로 eHSA에 결합할 수 있다는 것이 입증되었다. MST(Microscale Thermophoris)에 의한 FcRn과의 상호 작용은 eHSA가 혈류에서 긴 반감기를 가질 것을 검증한다.
- Issued Date
- 2021
- Awarded Date
- 2021. 8
- Type
- Dissertation
- Publisher
- 부경대학교
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 화학공학과
- Advisor
- Sung In Lim
- Table Of Contents
- ChapterI (Introduction) 1
1.1 Drug Delivery System 2
1.2. Albumin-based drug delivery 4
1.2.1. Albumin tertiary structure 5
1.2.2. Long half-life 6
1.2.3. Specificity 10
1.2.4. Albumin binding strategies 13
1.2.4.1. Covalent binding 13
1.2.4.2. Genetic fusion 14
1.2.4.3. Nanoformulation 15
1.3. Self-assembly domain 18
1.4. TNF-related apoptosis-inducing ligand (TRAIL) 20
1.5. Objective of this study 22
Chapter II (Materials and Methods) 24
2.1. Materials 25
2.2. Construction of plasmid and expression hosts 27
2.3. Protein expression 29
2.3.1. Transformation by heat shock 29
2.3.2. Optimization of recombinant protein expression 29
2.3.3. Batch of desired protein (production) 30
2.4. Protein Purification 32
2.4.1. Ni-NTA purification 32
2.4.2. FPLC 32
2.4.2.1. Buffer exchange 33
2.4.2.2. Anionic exchange chromatography 33
2.4.3. Calculation of the batch yield 34
2.5. Protein characterization 34
2.5.1. SDS-PAGE gel electrophoresis 35
2.5.1.1. Sample Preparation 35
2.5.1.2. Electrophoresis 36
2.5.1.3. Staining and destaining 36
2.5.2. Western blot analysis 36
2.5.2.1. Gel electrophoresis 37
2.5.2.2. Blotting to membrane 37
2.5.2.3. Detection 37
2.5.2.4. Imaging 38
2.6. Investigation of the interaction between two constructs 38
2.6.1. NATIVE-PAGE 38
2.6.2. HPLC-SEC 39
2.6.3. Isothermal Titration calorimetry (ITC) 39
2.6.3.1. Sample preparation 40
2.6.4. Microscale Thermophresis (MST) 40
2.6.4.1. Labeling 41
2.6.4.1.1. Analysis of labeling 41
2.6.4.2. Prepare samples 42
2.7. Cell assay 42
Chapter III (Results) 43
3.1. Design of the new albumin construct (Alboostumin) 44
3.2. Recombinant expression of the native and the engineered HSA 44
3.3. Result of protein purification 45
3.3.1. Ni affinity chromatography of the native and the engineered HSA 45
3.3.2. Ionic exchange chromatography of the native and the engineered HSA 47
3.3.3. Expression & purification of the GFP fused coil as payload 48
3.3.4. Expression & purification of the TRAIL 49
3.3.5. Expression & Purification of the monoTRAIL-coil as payload 50
3.3.6. Expression & purification of the 3TRAIL-coil as payload 51
3.3.7. Western Blot 52
3.4. Interaction Investigation 53
3.4.1. NATIVE-PAGE of the eHSA and the GFP-coil 53
3.4.2. HPLC-SEC of the eHSA and the GFP-coil 55
3.4.3. ITC of the eHSA and GFP-coil 56
3.4.4. NATIVEPAGE of the eHSA and monoTRAIL-coil 58
3.4.5. NATIVE-PAGE of the eHSA and 3TRAIL-coil 59
3.5. HPLC-SEC of the monoTRAIL-coil 60
3.6. FcRn binding (MST) 61
3.6.1. FcRn labeling 61
3.6.2. FcRn-HSA (WT) 62
3.6.3. FcRn-eHSA 63
3.6.4. FcRn-eHSA-payload 64
3.7. Cell assay 66
Chapter IV (Discussion) 69
References 79
- Degree
- Master
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