표제지
목차
Ⅰ. 서론 9
Ⅱ. 재료 및 방법 12
1. Chromosomal DNA from Pedobacter heparinus 12
2. Isolation of heparinase genes 12
3. Cloning and expression of heparinase I and III genes in E. coli 14
3.1. Restriction digestion of pET28a vector 14
3.2. Restriction digestion of heparinase genes 14
3.3. Ligation 15
3.4. Transformation 15
3.5. Plasmid DNA extraction and analysis 16
4. Heterologous protein expression and analysis 16
4.1. Expression of recombinant heparinases in E. coli 16
4.2. Identification of expressed recombinant heparinase I and III 17
5. Protein purification 20
5.1. Ni-IDA affinity chromatography 20
5.2. Dialysis 20
5.3. Western blot analysis 21
6. Denaturation and refolding 23
6.1. Denaturation and purification 23
6.2. Refolding 24
7. Enzymatic activity assay 25
Ⅲ. 결과 26
1. Characterization of recombinant heparinse I and III genes 26
1.1. Sequence analysis of recombinant heparinases genes 26
1.2. Heterologous expression analysis of recombinant heparinase I and III in E. coli 37
2. Identification of purified recombinant heparinase I and III 40
2.1. Analysis of his-tag purification 40
2.2. Analysis of soluble protein before and after dialysis 41
2.3. Analysis of denatured recombinant heparinase I 43
3. Enzymatic activity assay 46
3.1. Recombinant heparinase I 46
3.2. Recombinant heparinase III 48
Ⅳ. 고찰 50
Ⅴ. 참고 문헌 52
적요 55
Abstract 57