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

Ryu, Jina; Kim, Hyoju; Park, Hee Ho; Lee, Hong Jai; Park, Jae Young; Rhee, Won Jong

doi:10.1002/biot.201600040

Cited by 18 publications

(20 citation statements)

References 42 publications

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“…2, 5 and 11), and a straightforward correlation between the parameters of sgRNAs (e.g., GC content or target site location, Supplementary Table 2) and their gene activation efficiencies has not been established. Meanwhile, since dCas9-TV is prone to protein degradation due to its high sequence repetition, future studies will address the possibility whether genetic fusion of XTEN 26 , 30Kc19α 27 or other protein-stabilizing polypeptides to the N- or C-terminus of dCas9-TV may mitigate its degradation issue and further potentiate the dCas9-TV-mediated transcriptional activation.…”

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confidence: 99%

A potent Cas9-derived gene activator for plant and mammalian cells

et al. 2017

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Overexpression of complementary DNA (cDNA) represents the most commonly used gain-of-function approach for interrogating gene functions and for manipulating biological traits. However, this approach is challenging and inefficient for multigene expression due to increased labor for cloning, limited vector capacity, requirement of multiple promoters and terminators, and variable transgene expression levels. Synthetic transcriptional activators provide a promising alternative strategy for gene activation by tethering an autonomous transcription activation domain (TAD) to an intended gene promoter at endogenous genomic locus through a programmable DNA-binding module. Among the known custom DNA-binding modules, the nuclease-dead Streptococcus pyogenes Cas9 (dCas9) protein, which recognizes a specific DNA target through base pairing between a synthetic guide RNA (sgRNA) and DNA, outperforms zinc finger proteins (ZFPs) and transcription activator-like effectors (TALEs), both of which target through protein-DNA interactions1. Recently, three potent dCas9-based transcriptional activation systems, namely VPR, SAM, and Suntag, have been developed for animal cells2–6. However, an efficient dCas9-based transcriptional activation platform is still lacking for plant cells7–9. Here, we developed a new potent dCas9-TAD named dCas9-TV through plant cell-based screens, which confers far stronger transcriptional activation of a single or multiple target genes than the routinely used dCas9-VP64 activator in both plant and mammalian cells.

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confidence: 99%

A potent Cas9-derived gene activator for plant and mammalian cells

et al. 2017

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“…The 30Kc19 protein consists of six alpha-helixes of the N-terminal domain and 12 beta-strands of the C-terminal domain [ 85 ]. The CPP Pep-c19 is located in the α-helix domain [ 84 , 86 ]. Lee et al conducted a study to deliver β-galactosidase (β-gal) into cells using 30Kc19 protein nanoparticles [ 84 ].…”

Section: Types Of Proteins Used To Produce Protein Nanoparticlesmentioning

confidence: 99%

Protein-Based Nanoparticles as Drug Delivery Systems

et al. 2020

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Nanoparticles have been extensively used as carriers for the delivery of chemicals and biomolecular drugs, such as anticancer drugs and therapeutic proteins. Natural biomolecules, such as proteins, are an attractive alternative to synthetic polymers commonly used in nanoparticle formulation because of their safety. In general, protein nanoparticles offer many advantages, such as biocompatibility and biodegradability. Moreover, the preparation of protein nanoparticles and the corresponding encapsulation process involved mild conditions without the use of toxic chemicals or organic solvents. Protein nanoparticles can be generated using proteins, such as fibroins, albumin, gelatin, gliadine, legumin, 30Kc19, lipoprotein, and ferritin proteins, and are prepared through emulsion, electrospray, and desolvation methods. This review introduces the proteins used and methods used in generating protein nanoparticles and compares the corresponding advantages and disadvantages of each.

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“…The 30Kc19α and 30Kc19 genes were cloned and inserted into a pET-23a expression plasmid vector (Novagen, Madison, WI, USA) using the EcoRI and XhoI restriction enzyme sites, respectively, as described in previous studies [24,28]. The E. coli BL21 strain (DE3, Enzynomics, Daejeon, Korea) was then transformed with the recombinant plasmid and cultivated exponentially in Luria-Bertani (LB) broth medium until O.D.…”

Section: Plasmid Construction and Production Of Recombinant Protein I...mentioning

confidence: 99%

“…These findings suggest that the enhanced EPO productivity in recombinant CHO cells after the intracellular delivery of the 30Kc19 protein is due to the stabilization of mitochondrial enzyme complexes responsible for electron transport and ATP generation [27]. Meanwhile, other studies have demonstrated that the α-helix N-terminal domain of 30Kc19 (30Kc19α), dominantly expressed as a soluble form in E. coli, exhibits protein-stabilizing and cell-penetrating effects similar to or higher than the fulllength 30Kc19 protein [28,29]. However, the effect of the 30Kc19α protein on recombinant protein production in CHO cells compared to the effect of the full-length 30Kc19 protein has not yet been reported.…”

Section: Introductionmentioning

confidence: 98%

Recombinant Human Erythropoietin Production in Chinese Hamster Ovary Cells Is Enhanced by Supplementation of α-Helix Domain of 30Kc19 Protein

Cha

Park

2021

Applied Sciences

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The enhancement of recombinant therapeutic protein production in mammalian cell culture has been regarded as an important issue in the biopharmaceutical industry. Previous studies have reported that the addition of the recombinant 30Kc19 protein, a silkworm-derived plasma protein with simultaneous cell-penetrating and mitochondrial enzyme-stabilizing properties, can enhance the recombinant protein expression in Chinese hamster ovary (CHO) cell culture. Here, we produced an α-helix N-terminal domain of 30Kc19, called (30Kc19α), and investigated its effects on the production of human erythropoietin (EPO), a widely used therapeutic protein for the treatment of anemia, in recombinant CHO cell culture. Similar to the full-length 30Kc19, 30Kc19α was able to be mass-produced in a form of recombinant protein through an Escherichia coli expression system and delivered into EPO-producing CHO (EPO–CHO) cells. Supplementing the medium of EPO–CHO cell culture with 30Kc19α increased the intracellular NADPH/NADP+ ratio related to the flux of metabolic reducing power for protein biosynthesis, subsequently enhancing EPO production in serum-free culture. 30Kc19α is considered to have certain advantages in the downstream purification process of therapeutic protein production when it is used as a medium supplement due to its small size and low isoelectric point compared to the full-length 30Kc19. These results suggest that 30Kc19α has potential use for manufacturing biopharmaceutical proteins.

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

Cited by 18 publications

References 42 publications

A potent Cas9-derived gene activator for plant and mammalian cells

A potent Cas9-derived gene activator for plant and mammalian cells

Protein-Based Nanoparticles as Drug Delivery Systems

Recombinant Human Erythropoietin Production in Chinese Hamster Ovary Cells Is Enhanced by Supplementation of α-Helix Domain of 30Kc19 Protein

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