Complete Solubilization and Purification of Recombinant Human Growth Hormone Produced in Escherichia coli

Kim, Minji; Park, Hyun Soo; Seo, Kyung Hye; Yang, Hyo-Jin; Kim, Sook-Kyung; Choi, Jun-Hyuk

doi:10.1371/journal.pone.0056168

Cited by 52 publications

(40 citation statements)

References 35 publications

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“…reported lowering of the induction temperature significantly decreased the formation of somatropin inclusion bodies. They could solubilize most of the pelleted hGH under optimized culture conditions in periplasmic space (2427). …”

Section: Discussionmentioning

confidence: 99%

Twin arginine translocation system in secretory expression of recombinant human growth hormone

et al. 2016

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Recombinant protein production in E. coli has several advantages over other expression systems. Misfolding, inclusion body formation, and lack of eukaryotic post translational modification are the most disadvantages of this system. Exporting of correctly folded proteins to the outside of reductive cytoplasmic environment through twin-arginine system could help to pass these limiting steps. Two signal sequences, TorA and SufI are used at N-terminal of human growth hormone (hGH) bearing DsbA gene sequence at C-terminal to enhance folding. The synthetic cassettes including the signal sequence, hGH and DsbA were transformed into E. coli BL21 (DE3) to study the effect of signal sequence and DsbA chaperone on translocation and folding of the protein. The results confirmed using signal sequence at N-terminal of targeted protein and coexpression with DsbA could transport proteins to the periplasmic space and culture media compared to control groups. Although there is no protein band of somatropin in SDS-Page of culture media samples when using SufI as signaling sequence, the study demonstrated TorA signal sequence could transport the target protein to the culture media. However, there was a considerable amount of hGH in periplasmic space when using SufI compared to control.

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Section: Discussionmentioning

confidence: 99%

Twin arginine translocation system in secretory expression of recombinant human growth hormone

et al. 2016

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“…The reliability of the analysis was confirmed by at least two independent measurements under the same conditions. Detection was achieved by monitoring the UV absorbance at 280 nm (22). The purified assembly of the recombinant cellulosomes was confirmed by native PAGE analysis using a 12% polyacrylamide gel without SDS (Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

Unique Contribution of the Cell Wall-Binding Endoglucanase G to the Cellulolytic Complex in Clostridium cellulovorans

Jeon

Lee

Kim

et al. 2013

Appl Environ Microbiol

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bThe cellulosomes produced by Clostridium cellulovorans are organized by the specific interactions between the cohesins in the scaffolding proteins and the dockerins of the catalytic components. Using a cohesin biomarker, we identified a cellulosomal enzyme which belongs to the glycosyl hydrolase family 5 and has a domain of unknown function 291 (DUF291) with functions similar to those of the surface layer homology domain in C. cellulovorans. The purified endoglucanase G (EngG) had the highest synergistic degree with exoglucanase (ExgS) in the hydrolysis of crystalline cellulose (EngG/ExgS ratio ‫؍‬ 3:1; 1.71-fold). To measure the binding affinity of the dockerins in EngG for the cohesins of the main scaffolding protein, a competitive enzyme-linked interaction assay was performed. Competitors, such as ExgS, reduced the percentage of EngG that were bound to the cohesins to less than 20%; the results demonstrated that the cohesins prefer to bind to the common cellulosomal enzymes rather than to EngG. Additionally, in surface plasmon resonance analysis, the dockerin in EngG had a relatively weak affinity (30-to 123-fold) for cohesins compared with the other cellulosomal enzymes. In the cell wall affinity assay, EngG anchored to the cell surfaces of C. cellulovorans using its DUF291 domain. Immunofluorescence microscopy confirmed the cell surface display of the EngG complex. These results indicated that in C. cellulovorans, EngG assemble into both the cellulolytic complex and the cell wall complex to aid in the hydrolysis of cellulose substrates.

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“…Among the cell lines selected for analysis, Nb2-11, a rat lymphoma cell line, has been used in in vitro human growth hormone activity analyses as an alternative to classical rat weight gain assay (32,33). Among the cell lines selected for analysis, Nb2-11, a rat lymphoma cell line, has been used in in vitro human growth hormone activity analyses as an alternative to classical rat weight gain assay (32,33).…”

Section: Flow Cytometric Measurement Of Nb2-11 Cell Proliferationmentioning

confidence: 99%

Quantitative analysis of cell proliferation by a dye dilution assay: Application to cell lines and cocultures

et al. 2017

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Cell proliferation represents one of the most fundamental processes in biological systems, thus the quantitative analysis of cell proliferation is important in many biological applications such as drug screening, production of biologics, and assessment of cytotoxicity. Conventional proliferation assays mainly quantify cell number based on a calibration curve of a homogeneous cell population, and therefore are not applicable for the analysis of cocultured cells. Moreover, these assays measure cell proliferation indirectly, based on cellular metabolic activity or DNA content. To overcome these shortcomings, a dye dilution assay employing fluorescent cell tracking dyes that are retained within cells was applied and was diluted proportionally by subsequent cell divisions. Here, it was demonstrated that this assay could be implemented to quantitatively analyze the cell proliferation of different types of cell lines, and to concurrently analyze the proliferation of two types of cell lines in coculture by utilizing cell tracking dyes with different spectral characteristics. The mean division time estimated by the dye dilution assay is compared with the population doubling time obtained from conventional methods and values from literature. Additionally, dye transfer between cocultured cells was investigated and it was found that it is a characteristic of the cells rather than a characteristic of the dye. It was suggested that this method can be easily combined with other flow cytometric analyses of cellular properties, providing valuable information on cell status under diverse conditions. © 2017 International Society for Advancement of Cytometry.

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Complete Solubilization and Purification of Recombinant Human Growth Hormone Produced in Escherichia coli

Cited by 52 publications

References 35 publications

Twin arginine translocation system in secretory expression of recombinant human growth hormone

Twin arginine translocation system in secretory expression of recombinant human growth hormone

Unique Contribution of the Cell Wall-Binding Endoglucanase G to the Cellulolytic Complex in Clostridium cellulovorans

Quantitative analysis of cell proliferation by a dye dilution assay: Application to cell lines and cocultures

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