Heterologous Expression of Human GAD65 (IDDM Autoantigen) and Its Display on Protein Nanoparticles
- 발행기관 고려대학교 대학원
- 발행년도 2004
- 학위명 박사
- 학과 대학원:화공생명공학과
- 식별자(기타) DL:000014914304
- 서지제어번호 000045216082
초록/요약
Autoantibodies to human glutamic acid decarboxylase (GAD65) are found in most of type 1 diabetes mellitus patients many years before the appearance of clinical symptoms of the disease. It is known that a redundant reactivity of many GAD65-Ab positive sera to three major conformational (EP-1, EP-2, EP-3) and two dominant linear epitope clusters (amino acid 1-24 and 535-585) was observed in diabetes. Extensive studies on the structure, function, and therapeutic use of GAD65 have not been made. Owing to the lack of efficient expression system for providing a large amount of active recombinant GAD65. It has been reported that expression of recombinant GAD65 in Escherichia coli resulted in only insoluble and inactive proteins in immunochemical tests. Previous studies showed that the presence of ferritin heavy chain polypeptide at the amino terminus of the various human proteins significantly increased the cytoplasmic solubility of the recombinant ferritin hybrid. The GAD65 COOH-terminal fragments, i.e. GAD65448-585, GAD65487-585, and GAD65512-585 amino acid fragments, using FTN-H as N-terminus fusion expression partner in E. coli. All of recombinant fusion proteins (FTN-H::GAD65448-585, FTN-H::GAD65487-585, and FTN-H::GAD65512-585) formed spherical nanoparticles due probably to the self-assembly function of the fused ferritin heavy chain. The antigenic epitopes within GAD65448-585, GAD65487-585, and GAD65512-585 against type I diabetes marker (autoantibodies against GAD65) were localized at the surface of the spherical protein nanoparticles so that anti-GAD65 Ab could recognize them. Protein nanoparticles like FTN-H seem to provide distinct advantages over other inorganic nanoparticles (e.g. Au, Ag, CdSe, etc.) in that through the bacterial synthesis, the active capture probes can be located at the nanoparticle surface with constant orientation/conformation via covalent cross-linking without complex chemistry. Also it is possible for the protein nanoparticles to hav
more목차
ABSTRACT ⅰ
CONTENTS ⅲ
LIST OF TABLES ⅴ
LIST OF FIGURES ⅵ
Ⅰ. INTRODUCTION 1
1. Type 1 diabetes (Insulin-Dependent Diabetes Mellitus; IDDM) 1
2. Human glutamic acid decarboxylase 65 (GAD65) 2
2.1. Production of GAD65 4
2.2. Epitopes of GAD65 related to the formation of autoantibodies 5
3. Ferritin heavy chain (FTN-H) 8
4. Self-assembled protein nanoparticles - E. coli Dps & Listeria innocua ferritin 10
5. Protein chip 13
6. Objective 15
Ⅱ. MATERIALS AND METHODS 16
1. Construction of full length GAD65 expression vector 16
2. Construction of ferritin heavy-chain fusion GAD65 expression vector 16
3. Construction of ferritin fusion GAD65 fragment expression vectors 21
4. dps and ferritin gene isolation from bacterial strains and culture
conditions 25
4.1. Construction of E. coli Dps expression vector 25
4.2. Construction of L. innocua ferritin expression vector 25
5. Recombinant strains and gene expression 26
6. Electrophoresis and Western blot 27
7. Purification of recombinant proteins 27
7.1. Preparation of cell lysates 28
7.2. Preparation of the columns 28
7.3. Purification procedure 29
8. Transmission Electron Microscopy (TEM) 31
Ⅲ. RESULTS AND DISCUSSION 32
1. GAD65 whole gene Expression in E. coli 32
2. FTN-H::GAD65 Expression in E. coli. 35
3. GAD65-derived epitope fragments and fusion expression in E. coli 39
4. Self-assembled protein nanoparticles 46
5. Gene cloning and expression of E. coli Dps and L. innocua ferritin 50
Ⅳ. CONCLUSIONS 54
Ⅴ. REFERENCES 56
