title: Dimerization of 30Kc19 protein in the presence of amphiphilic moiety and importance of Cys-57 during cell penetration.
abstract: Recently, the recombinant 30Kc19 protein, originating from silkworm hemolymph of Bombyx mori has attracted attention due to its cell-penetrating property and potential application as a protein delivery system. However, this observation of penetration across cell membrane has raised questions concerning the interaction of the protein-lipid bilayer. Here, we report a dimerization propensity of the 30Kc19 protein in the presence of amphiphilic moieties; sodium dodecyl sulfate (SDS) or phospholipid. Native PAGE showed that the 30Kc19 monomer formed a dimer when SDS or phospholipid was present. In the glutathione-S-transferase (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 that the presence of cysteine at position 57 (Cys-57) is involved in dimerization of the 30Kc19 at the cell membrane during penetration. We anticipate application of the native 30Kc19 protein with high cell-penetrating efficiency for delivery of cargos to various cell types. The intracellular cargo delivery using the 30Kc19 protein is a non-virus derived (e.g. TAT) delivery method, which can open up new approaches for the delivery of therapeutics in bioindustries, such as pharma- and cosmeceuticals.
authors.affiliation: The School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea.
authors.forename: Hee Ho
authors.initials: HH
authors.lastname: Park
authors.forename: Youngsoo
authors.initials: Y
authors.lastname: Sohn
authors.forename: Ji Woo
authors.initials: JW
authors.lastname: Yeo
authors.forename: Ju Hyun
authors.initials: JH
authors.lastname: Park
authors.forename: Hong Jai
authors.initials: HJ
authors.lastname: Lee
authors.forename: Jina
authors.initials: J
authors.lastname: Ryu
authors.forename: Won Jong
authors.initials: WJ
authors.lastname: Rhee
authors.forename: Tai Hyun
authors.initials: TH
authors.lastname: Park
mesh_terms: D000818:Animals; D012831:Bombyx; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate
chemical_list: D057846:Cell-Penetrating Peptides; D019476:Insect Proteins; D010743:Phospholipids; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate
keywords: 30Kc19 protein; Dimerization; Penetration; Phospholipid; Sodium dodecyl sulfate