Identification and Characterization of Cell-penetrating Peptide from 30Kc19 Protein and Its Application to Protein and Gene Delivery : 30Kc19 단백질의 세포투과 펩타이드 동정과 특성 분석 및 단백질과 유전자 전달을 위한 응용

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공과대학 화학생물공학부
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서울대학교 대학원
30Kc19 proteindimerizationcell-penetrating peptide (CPP)siRNArecombinant protein productionin vivo delivery
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 research
the 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 moieties
SDS 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 penetration
due 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 CPP
VVNKLIRNNKMNC, 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.
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College of Engineering/Engineering Practice School (공과대학/대학원)Dept. of Chemical and Biological Engineering (화학생물공학부)Theses (Ph.D. / Sc.D._화학생물공학부)
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