Hong Jai Lee

Enzyme delivery using the 30Kc19 protein and human serum albumin nanoparticles

Nanoparticles have been widely used for delivering various chemical and biomolecular drugs, such as anti-cancer drugs and therapeutic proteins. Among nanoparticles, protein nanoparticles have advantages of non-cytotoxicity and biodegradability. In this study, a recombinant 30Kc19 protein was applied to human serum albumin (HSA) nanoparticles to enhance cellular uptake and stability of a nanoparticle cargo enzyme. The 30Kc19 protein, which originates from silkworm, has cell-penetrating and enzyme-stabilizing abilities. Therefore, 30Kc19-HSA nanoparticles were expected to enhance cellular uptake and stability of an enzyme loaded on the nanoparticles. Here, nanoparticles loaded with β-galactosidase were prepared using the desolvation method. The 30Kc19-HSA nanoparticles were uniformly spherical in shape, dispersed evenly in phosphate buffered saline and cell culture media, and released β-galactosidase in a sustained manner. The 30Kc19-HSA nanoparticles had negligible toxicity to animal cells and exhibited enhanced cellular uptake and intracellular stability of β-galactosidase in HeLa and HEK293 cells when compared with those of HSA nanoparticles. These results suggest that 30Kc19-HSA protein nanoparticles could be used as a versatile tool for drug delivery to various cells.

Microfluidic bead-based sensing platform for monitoring kinase activity

Protein kinases control cellular functions by regulating protein phosphorylation. Monitoring protein kinase activity is essential for medical diagnosis and drug screening. Here, we present a novel microfluidic device for performing simple and versatile protein kinase assays, which utilizes a microbead-based chemosensor. An automatic mix-and-measure technique was achieved using integrated pneumatic valves. After mixing each reagent for the kinase assay, the mixture was transferred to the sensing chamber. Then, phosphorylated and fluorescence-labeled peptides were captured and detected by the chemosensor. A fluorescence signal was observed depending on the presence of the kinase. Furthermore, activities of various kinases in the cell lysate and the inhibitory effect of specific chemicals on the kinases were monitored. These results indicate that chemosensor-based microfluidic chips can be developed as a versatile kinase assay system.

Dimerization of 30Kc19 protein in the presence of amphiphilic moiety and importance of Cys-57 during cell penetration

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.

Protein-stabilizing and cell-penetrating properties of α-helix domain of 30Kc19 protein

The protein‐stabilizing and cell‐penetrating activities of Bombyx mori 30Kc19 α‐helix domain (30Kc19α) are investigated. Recently, 30Kc19 protein has been studied extensively as it has both protein‐stabilizing and cell‐penetrating properties. However, it is unknown which part of 30Kc19 is responsible for those properties. 30Kc19 protein is composed of two distinct domains, an α‐helix N‐terminal domain (30Kc19α) and a β‐trefoil C‐terminal domain (30Kc19β). The authors construct and produce truncated forms of 30Kc19 to demonstrate their biological functions. Interestingly, 30Kc19α was shown to be responsible for both the protein‐stabilizing and cell‐penetrating properties of 30Kc19 protein. 30Kc19α shows even higher protein delivery activity than did whole 30Kc19 protein and has low cytotoxicity when added to cell culture medium. Therefore, based on its multifunctional properties, 30Kc19α can be developed as a novel candidate for a therapeutic protein carrier into various cells and tissues.

α-Galactosidase delivery using 30Kc19-human serum albumin nanoparticles for effective treatment of Fabry disease

Fabry disease is a genetic lysosomal storage disease caused by deficiency of α-galactosidase, the enzyme-degrading neutral glycosphingolipid that is transported to lysosome. Glycosphingolipid accumulation by this disease causes multi-organ dysfunction and premature death of the patient. Currently, enzyme replacement therapy (ERT) using recombinant α-galactosidase is the only treatment available for Fabry disease. To maximize the efficacy of treatment, enhancement of cellular delivery and enzyme stability is a challenge in ERT using α-galactosidase. In this study, protein nanoparticles using human serum albumin (HSA) and 30Kc19 protein, originating from silkworm, were used to enhance the delivery and intracellular α-galactosidase stability. 30Kc19-HSA nanoparticles loaded with the α-galactosidase were formed by desolvation method. 30Kc19-HSA nanoparticles had a uniform spherical shape and were well dispersed in cell culture media. 30Kc19-HSA nanoparticles had negligible toxicity to human cells. The nanoparticles exhibited enhanced cellular uptake and intracellular stability of delivered α-galactosidase in human foreskin fibroblast. Additionally, they showed enhanced globotriaosylceramide degradation in Fabry patients’ fibroblasts. It is expected that 30Kc19-HSA protein nanoparticles could be used as an effective tool for efficient delivery and enhanced stability of drugs.

Soluble expression and stability enhancement of transcription factors using 30Kc19 cell-penetrating protein

Transcription factors have been studied as an important drug candidate. Ever since the successful generation of induced pluripotent stem cells (iPSCs), there has been tremendous interest in reprogramming transcription factors. Because of the safety risks involved in a virus-based approach, many researchers have been trying to deliver transcription factors using nonintegrating materials. Thus, delivery of transcription factors produced as recombinant proteins in E. coli was proposed as an alternative method. However, the low level of soluble expression and instability of such recombinant proteins are potential barriers. We engineered a Bombyx mori 30Kc19 protein as a fusion partner for transcription factors to overcome those problems. We have previously reported that 30Kc19 protein can be produced as a soluble form in E. coli and has a cell-penetrating property and a protein-stabilizing effect. Transcription factors fused with 30Kc19 (Oct4-30Kc19, Sox2-30Kc19, c-Myc-30Kc19, L-Myc-30Kc19, and Klf4-30Kc19) were produced as recombinant proteins. Interestingly, Oct4 and L-Myc were expressed as a soluble form by conjugating with 30Kc19 protein, whereas Oct4 alone and L-Myc alone aggregated. The 30Kc19 protein also enhanced the stability of transcription factors both in vitro and in cells. In addition, 30Kc19-conjugated transcription factors showed rapid delivery into cells and transcriptional activity significantly increased. Overall, 30Kc19 protein conjugation simultaneously enhanced soluble expression, stability, and transcriptional activity of transcription factors. We propose that the conjugation with 30Kc19 protein is a novel approach to solve the technical bottleneck of gene regulation using transcription factors.