GIST Repository
College of Engineering
Department of Materials Science and Engineering
3. Theses(Master)
Albumin Variant expression in yeast and its application to therapeutics
- Abstract
- Therapeutic proteins are used to treat a variety of diseases due to their low side effects and low immunogenicity. However, many therapeutic protein candidates suffer from short half-lives, due to degradation by proteases and removal by kidney filtration. To address this issue, various studies have been conducted to increase the half-life by conjugating human serum albumin (HSA). Previous studies have successfully conjugated therapeutic proteins to albumin using the SpyTag/SpyCatcher (ST/SC) system, leading to the development of Albucatcher, a novel conjugation platform that increases the half-life while maintaining activity. However, despite its many advantages, the resulting Albucatcher Ver.1 required multiple steps to fabricate, which left challenges to overcome, including time, effort, and reduced yield. Albucatcher Ver.2 aims to effectively reduce the multiple steps required to conjugate albumin with SpyCatcher (SC) by creating an albumin genetically conjugated with SpyCatcher and introducing it into yeast. We have confirmed that Albucatcher Ver.2 successfully forms a Uox-albumin conjugate (Uox-HSA) with Urate Oxidase (Uox), the model protein used for previous conjugation, and maintains the same activity.
- Issued Date
- 2025
- Type
- Thesis
- Alternative Author(s)
- Moon GIseok
- Department
- 대학원 신소재공학부
- Advisor
- Kwon, Inchan
- Table Of Contents
- Abstract i․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․
List of contents ii․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․
List of figures v․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․
List of schemes v․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․
I. INTRODUCTION 1․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․
II. HSA and its variant Expression in Yeast Eukaryotic protein expression system
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1. Introduction ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․ 4
2. Experimental Materials & Methods ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․ 5
2.1. Materials ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․ 5
2.2. Construction of Plasmid ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․ 5
2.2.1. Point-mutation of pYD1 plasmid (Nco1 Insertion) ․․․․․․․․․․․ 5
2.2.2. pYD1-HSA Subcloning (For Display) ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․ 6
2.3. Yeast protein Expression and Display ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․ 6
2.4. Displayed HSA check with Fluorescent microscope ․․․․․․․․․․․․․․․ 7
3. Results and Discussion ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․ 7
3.1. Yeast Protein Display check with Fluorescent microscope
7․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․
4. Conclusion 9․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․
ii
III. Half-life extended HSA Variants (K573P), SpyCatcher inserted Albumin
(Albucatcher ver.2) expression and Albucatcher ver.2 Conjugation with Uricase
Oxidase (Uox) Therapeutic protein 10․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․
1. Introduction ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․ 10
2. Experimental Materials & Methods ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․ 12
2.1. Materials ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․ 12
2.2. Construction of Plasmid ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․ 12
2.2.1. Construction of pYD1-HSA (For Secretion) ․․․․․․․․․․․․․․․․․ 13
2.2.2. Half-life extended Albumin variant (K573P) ․․․․․․․․․․․․․․․․․ 13
2.2.3. Expression efficacy increasing sequence insertion ․․․․․ 13
2.2.4. Insert the SpyCatcher sequence to the N terminus and C
terminus of pYD1-HSA Plasmid ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․ 14
2.2.5. Introduce a linker sequence between HSA and SC ․․․ 15
2.2.6. Twin-strep-tag Insertion for Protein purification ․․․․․․ 15
2.3. Computational protein structure prediction using Alphafold 3
․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․ 16
2.4. Yeast protein expression and purification ․․․․․․․․․․․․․․․․․․․․․․․․ 17
2.5. Characterization of Expressed HSA-WT ․․․․․․․․․․․․․․․․․․․․․․․․․․․․ 17
2.6. Conjugation of Albucatcher ver.2 with Uox-ST ․․․․․․․․․․․․․․․ 18
2.7. SEC separation and Uox-HSA purification from mixed protein
sample ․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․ 18
2.8. Emzymatic Activity Assay of Uox-ST and Uox-HSA Conjugate
․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․ 18
3. Results and Discussion 19․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․
3.1. HSA-WT and HSA Variant Expression, Purification and
Characterization 19․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․
3.2. Uox-ST Expression and Purification 20․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․
iii
3.3. Uox-ST and Albucatcher ver.2 Conjugation and Purification
21․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․
3.4. In Vitro Uox-ST and Uox-HSA Activity Assay 24․․․․․․․․․․․․․․․․․․․․․
4. Conclusions 25․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․
Summary 26․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․
Reference 27․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․
Acknowledgment 29․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․․
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- Master
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- Department of Materials Science and Engineering > 3. Theses(Master)
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