30Kc19 단백질의 세포투과 펩타이드 동정과 특성 분석 및 단백질과 유전자 전달을 위한 응용

학위논문 (박사)-- 서울대학교 대학원 : 화학생물공학부, 2014. 8. 박태현.30Kc19 protein is a member of the 30K protein family, a similar structured protein found in hemolymph of Bombyx mori. These proteins have molecular weights of around 30 kDa, and 30Kc19 protein is the most abundant among 30K proteins (30Kc6, 30Kc12, 30Kc19, 30Kc21 and 30Kc23) in the hemolymph. Although the biological functions of the 30K proteins in silkworms have not been fully determined, several studies have recently examined their functional properties for 30Kc6 and 30Kc19. In previous studies, it was demonstrated that silkworm hemolymph and 30K proteins exhibit an anti-apoptotic effect in various cells by adding the protein to culture medium or by gene expression. 30K proteins also enhanced productions of recombinant erythropoietin, interferon-, and monoclonal antibody, as well as increasing glycosylation, cell growth, and viability in various cells, and also had enzyme-stabilizing effects. A recent study has shown that 30Kc19 protein has a cell-penetrating property when supplemented to the culture medium. Therefore, 30Kc19 protein is a very unique multi-functional protein, and can be applied for the delivery of therapeutic proteins, including enzymes, as it can penetrate cell membrane as well as stabilizing cargo proteins. The dimerization propensity of the 30Kc19 in the presence of amphiphiles led to the objective of this researchthe investigation on the mechanism of internalization of the 30Kc19 and the minimal effective partial sequence of the parent protein that is necessary for the cell-penetrating to occur and delivery of cargos into cells. First, dimerization propensity of the 30Kc19 protein in the presence of amphiphilic moietiesSDS and phospholipid was investigated. Native PAGE result showed that the 30Kc19 monomer formed a dimer when SDS or phospholipid was present. From the GST pull-down assay, supplementation of the 30Kc19 protein to mammalian cell culture medium showed dimerization and penetrationdue to phospholipids at the cell membrane, the main components of the lipid bilayer. Mutagenesis was performed, and penetration was observed by 30Kc19 C76A and not 30Kc19 C57A, which meant Cys-57 is involved in dimerization of the 30Kc19 at the cell membrane during penetration. Then explored how the cell-penetrating 30Kc19 protein is related with phospholipids, the main cell membrane components, and elucidate the mechanism of entry of the 30Kc19 protein into animal cells for use in protein delivery system. Based on the cell penetrating mechanism and cargo-delivery ability, hypothetical presence of cell-penetrating peptide (CPP) was assumed and endeavored in the identification of a CPP of the 30Kc19 protein originating from the silkworm. For the practical use in delivery of cell-impermeable cargo molecules, it is necessary to find a cell-penetrating domain like other cell-penetrating proteins that can efficiently deliver cargo molecules into cells. A domain was selected as the most probable candidate for CPP and several CPP candidates were tested for cell-penetrating property. From this, a new CPPVVNKLIRNNKMNC, from 30Kc19 protein (Pep-c19) was identified and demonstrated that 30Kc19 exhibited a cell-penetrating property due to the presence of a cell-penetrating peptide at 45-57. Efficiency and toxicity of this CPP was investigated in comparison with its original protein, 30Kc19, both in vitro and in vivo and showed its superiority over its parent protein in terms of efficiency. Pep-c19 is a cell-penetrating peptide derived from the first cell-penetrating protein in insect hemolymph. Through this finding, anticipate in finding other cell-penetrating peptides from other proteins sourced from insects that have similar properties to Pep-c19. For therapeutic application, non-covalent approach for the delivery of siRNA. The results showed that Pep-c19 was able to deliver siRNAs and have gene silencing effect along with 11R, other widely recognized CPP. The 30Kc19 protein and Pep-c19 are a non-virus derived (e.g. TAT) and non-cytotoxic cell-penetrating protein/peptide. This study may open up new approaches and provide beneficial effects for the delivery of therapeutics in bioindustries, such as pharma- and cosmeceuticals.Contents Chapter 1. Research background and objectives 1 Chapter 2. Literature review 5 2.1 Cell-penetrating peptides (CPPs) 6 2. 1. 1 Terminology, classification, and structure of CPPs 6 2. 1. 2 Mechanism of penetration 8 2. 1. 3 Penetration of CPPs in vitro and in vivo 11 2. 1. 4 Penetration of CPPs with cargos 14 2. 1. 5 Toxicity of CPPs 16 2. 1. 6 Detecting internalization of CPPs 19 Chapter 3. Experimental procedures 21 3. 1. Construction of plasmid 22 3. 2. Production and purification of proteins and peptides 23 3. 3. Reducing SDS-PAGE, non-reducing SDS-PAGE, and native PAGE 23 3. 4. Cell culture 25 3. 5. Immunoblot analysis 25 3. 6. Quantitative internalization analysis 26 3. 7. GST pull-down assay 27 3. 8. Construction of HEK 293 stable cell line expressing EGFP 27 3. 9. Formation of CPP/siRNA complex 28 3. 10. Gel shift/retardation assay for CPP/siRNA complex 28 3. 11. Cell viability assay 28 3. 12. Immunocytochemistry and live cell analysis 29 3. 13. Inhibitors/effectors of endocytosis 30 3. 14. In vivo penetration of Pep-c19 30 3. 15. In vivo toxicity analysis 31 Chapter 4. Dimerization of the 30Kc19 protein in the presence of amphiphilic moieties and importance of Cys-57 during cell penetration 32 4. 1. Introduction 33 4. 2. Cell-penetrating property of the 30Kc19 protein 35 4. 3. Dimerization of the 30Kc19 protein is promoted by SDS 35 4. 4. Dimerization of the 30Kc19 protein is promoted by phospholipid 39 4. 5. Dimerization of the 30Kc19 during cell penetration 40 4. 6. Importance of the 30Kc19 Cys-57 for cell penetration 44 4. 7. Intracellular penetration in the presence of inhibitors of endocytosis 47 4. 8. Intracellular cargo delivery using the 30Kc19 protein 47 4. 9. Penetration mechanism of the 30Kc19 protein 49 4. 10. Conclusions 53 Chapter 5. Prediction and identification of cell-penetrating peptide of the 30Kc19 protein (Pep-c19) 54 5. 1. Introduction 55 5. 2. Presence of CPP in the 30Kc19 protein 56 5. 3. Identification of the 30Kc19 CPP (Pep-c19) 60 5. 4. Intracellular penetration in the presence of inhibitors of endocytosis 63 5. 5. Comparison of Pep-c19 with other cell-penetrating peptides 65 5. 6. Conclusions 67 Chapter 6. In vitro and in vivo protein delivery system using cell-penetrating peptide of the 30Kc19 protein (Pep-c19) 68 6. 1. Introduction 69 6. 2. In vitro protein delivery of protein-conjugated Pep-c19 70 6. 3. In vivo protein delivery of protein-conjugated Pep-c19 74 6. 4. Toxicity test for the long-term administration of Pep-c19 75 6. 5. Conclusions 80 Chapter 7. In vitro siRNA delivery system using cell-penetrating peptide of the 30Kc19 protein (Pep-c19) 81 7. 1. Introduction 82 7. 2. Formation of non-covalent CPP/siRNA complex 85 7. 3. Internalization of non-covalent CPP/siRNA complex in HeLa cells 87 7. 4. Effect of CPP/siRNA complex on the fluorescence of HEK 293-EGFP cells 87 7. 5. Cytotoxicity of CPP/siRNA complex 92 7. 6 Conclusions 95 Chapter 8. Overall discussion and further suggestions 96 8. 1. Overall discussion 97 8. 2. Conclusion and further suggestions 103 Bibliography 107 Abstract 125   List of Figures Figure 2.1.2 Mechanism of entry 13 Figure 2.1.4 Intracellular delivery of cargos using CPP 15 Figure 4.1 Structure of the 30Kc19 protein 34 Figure 4.2 Cell-penetrating property of the 30Kc19 protein 36 Figure 4.3 Dimerization of the 30Kc19 protein is promoted by SDS 38 Figure 4.4 Dimerization of the 30Kc19 protein is promoted by phospholipid41 Figure 4.5 Dimerization of 30Kc19 during cell penetration 43 Figure 4.6 Importance of 30Kc19 Cys-57 for cell penetration 46 Figure 4.7 Intracellular penetration in the presence of inhibitors of endocytosis 48 Figure 4.8 Intracellular cargo delivery using the 30Kc19 protein 50 Figure 4.9 Penetration mechanism of the 30Kc19 protein 52 Figure 5.1 Application of cell-penetrating peptides 57 Figure 5.2 Potential presence of cell-penetrating peptide in the 30Kc19 protein 59 Figure 5.3 Identification of Pep-c19 62 Figure 5.4 Intracellular penetration in the presence of inhibitors of endocytosis 64 Figure 6.1 Intraperitoneal injection of Pep-c19 and organ preparation from mice for in vivo delivery 71 Figure 6.2 Delivery of GFP into the cells by Pep-c19 73 Figure 6.3.1 In vivo delivery of GFP into various tissues by Pep-c19 76 Figure 6.3.2 In vivo delivery of GFP into various tissues by Pep-c19 77 Figure 6.4 Toxicity test for the long-term administration of Pep-c19 79 Figure 7.1 Principle of RNAi 83 Figure 7.2 Gel shift/retardation assay for CPP to form complexes with siRNA 86 Figure 7.3 Internalization of non-covalent CPP/siRNA-Cy3 complex 88 Figure 7.4.1 Gene silencing effect of non-covalent CPP-EGFP siRNAs 90 Figure 7.4.2 Gene silencing effect of non-covalent CPP-EGFP siRNAs 91 Figure 7.5 Cytotoxicity of non-covalent CPP-EGFP siRNAs 94 List of Tables Table 2.1.1 Examples of cell-penetrating peptides (CPPs) 9 Table 2.1.2 Classification of CPPs based on their mechanism. 12 Table 5.5 Comparison of Pep-c19 with other cell-penetrating peptides. 66Docto