Expression and Secretion of Cyan Fluorescent Protein (CFP) in B. subtilis using the Chitinase Promoter from Bacillus pumilus SG2

Shali, Abbas; Rigi, Garshasb; Pornour, Majid; Ahmadian, Gholamreza

doi:10.18869/acadpub.ibj.21.4.240

Cited by 5 publications

(2 citation statements)

References 33 publications

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“…The secretory pathways of Sec and twin-arginine translocation (Tat) pathways function in parallel to transport proteins across the cytoplasmic membranes of prokaryotes as unfolded and folded, respectively (Palmer et al 2012). So far, several proteins have been secreted extracellularly with this strategy using modi ed forms of signal peptides [23][24][25][26][27][28][29][30][31][32][33].…”

Section: Discussionmentioning

confidence: 99%

Optimization of Expression, Purification and Secretion of Functional Recombinant Human Growth Hormone in Prokaryotic Hosts Using Modified Staphylococcal Protein A Signal Peptide

Rigi

Rostami

Ghomi

et al. 2021

Preprint

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Background: Human Growth Hormone (hGH) is a glycoprotein released from the pituitary gland. Due to the wide range of effects in humans, any disruption in hGH secretion could have serious consequences. This highlights the clinical importance of hGH production in the treatment of different diseases associated with a deficiency of this hormone. The production of recombinant mature hormone in suitable hosts and secretion of this therapeutic protein into the extracellular space can be considered as one of the best cost-effective approaches not only to obtain the active form of the protein but also endotoxin-free preparation. Since the natural growth hormone signal peptide is of eukaryotic origin and is not detectable by any of the E. coli secretory systems, including Sec and Tat, and is therefore unable to secrete hGH in the prokaryotic systems, designing a new and efficient signal peptide is essential to direct hGh to the extracellular space. Results: In this study, using a combination of the bioinformatics design and molecular genetics, the protein A signal peptide from Staphylococcus aureus was modified, redesigned and then fused to the mature hGH coding region. The recombinant hGH was then expressed in E. coli and successfully secreted to the medium through the Sec pathway. Secretion of the hGH into the medium was verified using SDS-PAGE and western blot analysis. Recombinant hGH was then expressed in E. coli and successfully secreted into cell culture medium via the Sec pathway. The secretion of hGH into the extracellular medium was confirmed by SDS-PAGE and Western blot analysis. Furthermore, the addition of glycine was shown to improve hGH secretion onto the culture medium. Equations for determining the optimal conditions were also determined. Functional hGH analysis using an ELISA-based method confirmed that the ratio of the active form of secreted hGH to the inactive form in the periplasm is higher than this ratio in the cytoplasm.Conclusions: Since the native signal protein peptide of S. aureus protein A was not able to deliver hGH to the extracellular space, it was modified using bioinformatics tools and fused to the n-terminal region of hGh to show that the redesigned signal peptide was functional.

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Section: Discussionmentioning

confidence: 99%

Optimization of Expression, Purification and Secretion of Functional Recombinant Human Growth Hormone in Prokaryotic Hosts Using Modified Staphylococcal Protein A Signal Peptide

Rigi

Rostami

Ghomi

et al. 2021

Preprint

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“…Bacillus produces siderophores in order to adhere to the surface of nonbiological biofilm formation, which makes the Bacillus secretes broad-spectrum resistance compounds to a certain extent and increases the spectrum resistance of plants (Höfte and Bakker, 2007). Asaka and Shoda, (1996) reported that B. subtilis secreted antibiotics iturin A and surfactin for controlling plant diseases and enhancing the expression of the chitinase promoter (Shali et al, 2017). The high chitinase activity might have resulted in lyses of invading fungal pathogens (Fernando et al, 2007).…”

Section: Bioagentsmentioning

confidence: 99%

Effect of Some Bioagents on Cladosporium cucumerinum Causing Cladosporium Leaf Spot: A New Challenge in Watermelon Production in Egypt.

Farag

Aref

2019

Egyptian Journal of Phytopathology

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even isolates of bioagents, Trichoderma harzianum, Pseudomonas fluorescens, Bacillus subtilis, Anabena oryzae, Nostoc muscorum, Chaetomium globosum and Gliocladium catenulatum were used to control cladosporium leaf spot. Nostoc muscorum mixed with Pseudomonas fluorescens successfully reduced the growth and spore germination of Cladosporium cucumerinum. The potentially of the bioagents significantly reduced the cladosporium leaf spot disease incidence and inhibiting its progress when used each alone or as mixtures under field conditions. The bioagents generally improved the watermelon plants and significantly increased the plant height, number of branches, fruit numbers and fruits weight due to treatment with the tested bioagents either each alone or in mixture compared to the untreated control.

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Optimization of expression, purification and secretion of functional recombinant human growth hormone in Escherichia coli using modified staphylococcal protein a signal peptide

et al. 2021

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Background Human Growth Hormone (hGH) is a glycoprotein released from the pituitary gland. Due to the wide range of effects in humans, any disruption in hGH secretion could have serious consequences. This highlights the clinical importance of hGH production in the treatment of different diseases associated with a deficiency of this hormone. The production of recombinant mature hormone in suitable hosts and secretion of this therapeutic protein into the extracellular space can be considered as one of the best cost-effective approaches not only to obtain the active form of the protein but also endotoxin-free preparation. Since the natural growth hormone signal peptide is of eukaryotic origin and is not detectable by any of the Escherichia coli secretory systems, including Sec and Tat, and is therefore unable to secrete hGH in the prokaryotic systems, designing a new and efficient signal peptide is essential to direct hGh to the extracellular space. Results In this study, using a combination of the bioinformatics design and molecular genetics, the protein A signal peptide from Staphylococcus aureus was modified, redesigned and then fused to the mature hGH coding region. The recombinant hGH was then expressed in E. coli and successfully secreted to the medium through the Sec pathway. Secretion of the hGH into the medium was verified using SDS-PAGE and western blot analysis. Recombinant hGH was then expressed in E. coli and successfully secreted into cell culture medium via the Sec pathway. The secretion of hGH into the extracellular medium was confirmed by SDS-PAGE and Western blot analysis. Furthermore, the addition of glycine was shown to improve hGH secretion onto the culture medium. Equations for determining the optimal conditions were also determined. Functional hGH analysis using an ELISA-based method confirmed that the ratio of the active form of secreted hGH to the inactive form in the periplasm is higher than this ratio in the cytoplasm. Conclusions Since the native signal protein peptide of S. aureus protein A was not able to deliver hGH to the extracellular space, it was modified using bioinformatics tools and fused to the n-terminal region of hGh to show that the redesigned signal peptide was functional.

show abstract

Expression and Secretion of Cyan Fluorescent Protein (CFP) in B. subtilis using the Chitinase Promoter from Bacillus pumilus SG2

Cited by 5 publications

References 33 publications

Optimization of Expression, Purification and Secretion of Functional Recombinant Human Growth Hormone in Prokaryotic Hosts Using Modified Staphylococcal Protein A Signal Peptide

Optimization of Expression, Purification and Secretion of Functional Recombinant Human Growth Hormone in Prokaryotic Hosts Using Modified Staphylococcal Protein A Signal Peptide

Effect of Some Bioagents on Cladosporium cucumerinum Causing Cladosporium Leaf Spot: A New Challenge in Watermelon Production in Egypt.

Optimization of expression, purification and secretion of functional recombinant human growth hormone in Escherichia coli using modified staphylococcal protein a signal peptide

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