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

Abstract 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.

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

10.21203/rs.3.rs-257852/v1 ◽

2021 ◽

Author(s):

Garshasb Rigi ◽

Amin Rostami ◽

Habib Ghomi ◽

Gholamreza Ahmadian ◽

Vasiqe Sadat Mirbagheri ◽

...

Keyword(s):

Growth Hormone ◽

Western Blot ◽

Signal Peptide ◽

Extracellular Space ◽

Culture Medium ◽

Active Form ◽

Protein A ◽

Human Growth ◽

E Coli ◽

Sds Page

Abstract 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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Use of Bacteriocin Release Protein in E. Coli for Excretion of Human Growth Hormone into the Culture Medium

Nature Biotechnology ◽

10.1038/nbt0389-267 ◽

1989 ◽

Vol 7 (3) ◽

pp. 267-271 ◽

Cited By ~ 38

Author(s):

Hansen M. Hsiung ◽

Amanda Cantrell ◽

Joen Luirink ◽

Bauke Oudega ◽

Angelo J. Veros ◽

...

Keyword(s):

Growth Hormone ◽

Human Growth Hormone ◽

Culture Medium ◽

Human Growth ◽

E Coli ◽

Bacteriocin Release Protein

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Escherichia coli BL21(DE3) expression system using TorA signal peptide for Recombinant Human Albumin (rHA) secretion

International Journal of Research in Pharmaceutical Sciences ◽

10.26452/ijrps.v10i4.1640 ◽

2019 ◽

Vol 10 (4) ◽

pp. 3319-3324 ◽

Cited By ~ 1

Author(s):

Iman P. Maksum ◽

Astri Lestari ◽

Retna P. Fauzia ◽

Saadah D. Rachman ◽

Ukun M.S. Soedjanaatmadja

Keyword(s):

Escherichia Coli ◽

Molecular Weight ◽

Signal Peptide ◽

Recombinant Dna ◽

Expression System ◽

Foreign Protein ◽

Competent Cell ◽

Promising Alternative ◽

E Coli ◽

Sds Page

Human serum albumin (HSA) is the most abundant protein in blood plasm. This protein consisted of 585 amino acids with a molecular weight of 66 kDa and 17 disulfide bonds. HSA obtained from conventional technique allow viral or prion contamination. For that reason, recombinant DNA technology becomes a promising alternative. Because of its well-known genetic, simplicity, and capacity to accommodate many foreign protein, Escherichia coli remains the most widely used in the production of recombinant proteins. But, overproduction of protein may lead to the formation of inclusion bodies and proteolytic degradation. These problems can be overcome by using protease-deficient strain and protein secretion into periplasmic space. The objective of this research is to secrete recombinant HA on E. coli BL21(DE3) using TorA signal peptide and proved using SDS-PAGE. This research method begins with the preparation of competent cell and transformation of E. coli BL21(DE3), expression of recombinant HA in E. coli BL21(DE3), and characterization of expression result by using SDS-PAGE. The result of this study was rHSA can be secreted into extracellular medium using TorA signal peptide with a molecular weight of ± 66.5 kDa.

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Production and bioactivity test of recombinant protein common carp growth hormone

Jurnal Akuakultur Indonesia ◽

10.19027/jai.10.44-50 ◽

2011 ◽

Vol 10 (1) ◽

pp. 44

Author(s):

Deny Sapto Chondro Utomo ◽

. Alimuddin ◽

Agus Oman Sudrajat ◽

Irvan Faizal

Keyword(s):

Escherichia Coli ◽

Growth Hormone ◽

Body Weight ◽

Recombinant Protein ◽

Common Carp ◽

Cyprinus Carpio ◽

Bacterial Cells ◽

Recombinant Growth Hormone ◽

E Coli ◽

Sds Page

This study aimed to produce recombinant growth hormone (rGH) protein of common carp (Cyprinus carpio) and evaluate its bioactivity. DNA fragment encoding mature GH protein of common carp (mCcGH) was amplified by polymerase chain reaction (PCR) method and PCR products were then ligated into pCold I to generate pCold I-mCcGH protein expression vector. Escherichia coli BL21 (DE3) harboring pCold I-mCcGH was cultured in the 2xYT medium at 15 °C for 24 hours and protein production was induced by isopropyl-beta-thio galactopyranoside (IPTG). The inclusion bodies containing rGH protein from E. coli transformants were isolated by sonication method and analyzed by SDS-PAGE. The result showed that rGH with molecular weight of about 25 kDa was obtained. Common carp juveniles with average body weight of 5.2±0.4 g were intramuscularly injected once a week for 4 weeks with rGH protein solution from 1 μg bacterial cells per gram fish body weight. The result showed that juveniles fish injected with rGH grew 106.56% higher than control. This result indicated that rGH produced in E. coli BL21 possessed biological activity and it may be useful to improve growth of aquaculture species. Key words: growth hormone, recombinant protein, common carp ABSTRAK Penelitian ini bertujuan menghasilkan protein rekombinan hormon pertumbuhan (growth hormone, GH) dari ikan mas (Cyprinus carpio) dan menguji bioaktivitasnya. Fragmen DNA penyandi protein matang (mature) GH ikan mas (mCcGH) diamplifikasi dengan menggunakan metode PCR dan hasilnya kemudian diligasi ke dalam pCold-I untuk menghasilkan konstruksi vektor ekspresi pCold-I-mCcGH. Plasmid pCold-I-mCcGH ditransformasi ke bakteri Escherichia coli BL21 (DE3), dikultur dalam media 2xYT cair pada suhu 15°C selama 24 jam dan produksi protein diinduksi dengan menggunakan isopropyl-beta-thio galactopyranoside (IPTG). Badan inklusi yang mengandung protein rekombinan GH (rGH) dari bakteri E. coli transforman diisolasi menggunakan metode sonikasi dan dianalisis dengan menggunakan SDS-PAGE. Hasil penelitian menunjukkan bahwa rGH dengan bobot molekul sekitar 25 kDa berhasil diproduksi. Benih ikan mas dengan bobot rata-rata 5,15±0,4 g diinjeksi secara intramuskular satu kali per minggu selama 4 minggu dengan larutan rGH hasil ekstraksi dari 1 µg pelet bakteri/g bobot ikan. Benih yang disuntik dengan rGH tumbuh sekitar 100% lebih cepat bila dibandingkan dengan kontrol yang tidak diinjeksi rGH. Hasil ini mengindikasikan bahwa rGH yang diproduksi dalam bakteri E. coli memiliki bioaktivitas dan dapat bermanfaat untuk memacu pertumbuhan spesies ikan-ikan budidaya. Kata kunci: hormon pertumbuhan, protein rekombinan, ikan mas

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Soluble Expression, One-Step Purification and Characterization of Recombinant Human Growth Hormone Fused with ompA3 in Escherichia coli

Protein and Peptide Letters ◽

10.2174/0929866527666201110123426 ◽

2020 ◽

Vol 27 ◽

Author(s):

Zhen-Ru Zhou ◽

Wei Huang ◽

Kang-Jia Liu ◽

Fo-Lan Lin ◽

Xiao-Lu Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Growth Hormone ◽

Human Growth Hormone ◽

Recombinant Expression ◽

Recombinant Human Growth Hormone ◽

Human Growth ◽

Soluble Expression ◽

Hormone Deficiency ◽

Promoting Effect ◽

E Coli

Background: Human growth hormone (hGH) is the first recombinant protein approved for the treatment of human growth hormone deficiency. However, expression in inclusion bodies and low expression levels are enormous challenges for heterologous expression of hGH in Escherichia coli. Objective: To increase the soluble expression of recombinant hGH with correct folding in E. coli. Method: We constructed a new recombinant expression plasmid containing the coding sequence of the outer membrane protein A (ompA3) which was used for the expression in Transetta (DE3) E. coli. In order to simplify the purification process and cleavage of recombinant proteins, the fusion sequence should contain hexahistidine-tag (His6) and enterokinase recognition sites(D4K). The effect of different expression conditions on recombinant hGH expression was optimized in flask cultivations. Furthermore, the periplasmic solution containing soluble hGH was purified by Ni-NTA affinity chromatography. Circular dichroism (CD), western blot and mass spectrometry analyses were used to characterize the protein. Moreover, the growth-promoting effect of the purified hGH was also evaluated by cell proliferation assay. Results: High-level expression (800 g/mL) was achieved by induction with 0.5 mM IPTG at 30 ºC for 10 hours. The purity of hGH was over 90%. The immunological activity, secondary structure and molecular weight of the purified hGH were consistent with native hGH. The purified hGH was found to promote the growth of MC3T3-E1 cells, and was found to show the highest activity at a concentration of 100 ng/mL. Conclusion: Our research provides a feasible and convenient method for the soluble expression of recombinant hGH in E. coli, and may lay a foundation for the production and application of hGH in the industry.

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In Silico Investigation of Signal Peptide Sequences to Enhance Secretion of CD44 Nanobodies Expressed in Escherichia coli

Current Pharmaceutical Biotechnology ◽

10.2174/1389201021666201012162904 ◽

2020 ◽

Vol 21 ◽

Author(s):

Soudabeh Kavousipour ◽

Shiva Mohammadi ◽

Ebrahim Eftekhar ◽

Mahdi Barazesh ◽

Mohammad Hossein Morowvat

Keyword(s):

Escherichia Coli ◽

Recombinant Proteins ◽

Signal Peptide ◽

In Silico ◽

Extracellular Space ◽

Peptide Sequence ◽

Signal Peptides ◽

Signal Peptide Sequence ◽

E Coli ◽

Peptide Database

Background: The selection of a suitable signal peptide that can direct recombinant proteins from the cytoplasm to the extracellular space is an important criterion affecting the production of recombinant proteins in Escherichia coli, a widely used host. Nanobodies are currently attracting the attention of scientists as antibody alternatives due to their specific properties and feasibility of production in E. coli. Objective: CD44 nanobodies constitute a potent therapeutic agent that can block CD44/HA interaction in cancer and inflammatory diseases. This molecule may also function as a drug against cancer cells and has been produced previously in E. coli without a signal peptide sequence. The goal of this project was to find a suitable signal peptide to direct CD44 nanobody extracellular secretion in E. coli that will potentially lead to optimization of experimental methods and facilitate downstream steps such as purification. Methods: We analyzed 40 E. coli derived signal peptides retrieved from the Signal Peptide database and selected the best candidate signal peptides according to relevant criteria including signal peptide probability, stability, and physicochemical features, which were evaluated using signalP software version 4.1 and the ProtParam tool, respectively. Results: In this in silico study, suitable candidate signal peptide(s) for CD44 nanobody secretory expression were identified. CSGA, TRBC, YTFQ, NIKA, and DGAL were selected as appropriate signal peptides with acceptable D-scores, and appropriate physicochemical and structural properties. Following further analysis, TRBC was selected as the best signal peptide to direct CD44 nanobody expression to the extracellular space of E. coli. Conclusion: The selected signal peptide, TRBC is the most suitable to promote high level secretory production of CD44 nanobodies in E. coli and potentially will be useful for scaling up CD44 nanobody production in experimental research as well as in other CD44 nanobody applications. However, experimental work is needed to confirm the data.

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Suitable Signal Peptides for Secretory Production of Recombinant Granulocyte Colony Stimulating Factor in Escherichia coli

Recent Patents on Biotechnology ◽

10.2174/1872208314999200730115018 ◽

2020 ◽

Vol 14 (4) ◽

pp. 269-282

Author(s):

Sadra S. Tehrani ◽

Golnaz Goodarzi ◽

Mohsen Naghizadeh ◽

Seyyed H. Khatami ◽

Ahmad Movahedpour ◽

...

Keyword(s):

Escherichia Coli ◽

Signal Peptide ◽

Fusion Proteins ◽

Inclusion Bodies ◽

Clinical Aspects ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

E Coli ◽

Secretory Production ◽

Stimulating Factor

Background: Granulocyte colony-stimulating factor (G-CSF) expressed in engineered Escherichia coli (E. coli) as a recombinant protein is utilized as an adjunct to chemotherapy for improving neutropenia. Recombinant proteins overexpression may lead to the creation of inclusion bodies whose recovery is a tedious and costly process. To overcome the problem of inclusion bodies, secretory production might be used. To achieve a mature secretory protein product, suitable signal peptide (SP) selection is a vital step. Objective: In the present study, we aimed at in silico evaluation of proper SPs for secretory production of recombinant G-CSF in E. coli. Methods: Signal peptide website and UniProt were used to collect the SPs and G-CSF sequences. Then, SignalP were utilized in order to predict the SPs and location of their cleavage site. Physicochemical features and solubility were investigated by ProtParam and Protein-sol tools. Fusion proteins sub-cellular localization was predicted by ProtCompB. Results: LPP, ELBP, TSH, HST3, ELBH, AIDA and PET were excluded according to SignalP. The highest aliphatic index belonged to OMPC, TORT and THIB and PPA. Also, the highest GRAVY belonged to OMPC, ELAP, TORT, BLAT, THIB, and PSPE. Furthermore, G-CSF fused with all SPs were predicted as soluble fusion proteins except three SPs. Finally, we found OMPT, OMPF, PHOE, LAMB, SAT, and OMPP can translocate G-CSF into extracellular space. Conclusion: Six SPs were suitable for translocating G-CSF into the extracellular media. Although growing data indicate that the bioinformatics approaches can improve the precision and accuracy of studies, further experimental investigations and recent patents explaining several inventions associated to the clinical aspects of SPs for secretory production of recombinant GCSF in E. coli are required for final validation.

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High-level production and one-step purification of biologically active human growth hormone in Escherichia coli

Gene ◽

10.1016/0378-1119(95)00525-b ◽

1995 ◽

Vol 165 (2) ◽

pp. 303-306 ◽

Cited By ~ 29

Author(s):

Reema Mukhija ◽

Prithy Rupa ◽

Devika Pillai ◽

Lalit C. Garg

Keyword(s):

Escherichia Coli ◽

Growth Hormone ◽

Human Growth Hormone ◽

Human Growth ◽

Biologically Active ◽

One Step ◽

High Level ◽

Level Production

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Effective production of human growth factors in Escherichia coli by fusing with small protein 6HFh8

Microbial Cell Factories ◽

10.1186/s12934-020-01502-1 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Young Su Kim ◽

Hye-Jeong Lee ◽

Man-ho Han ◽

Nam-kyung Yoon ◽

Yeu-chun Kim ◽

...

Keyword(s):

Escherichia Coli ◽

Growth Factors ◽

Growth Factor ◽

Fusion Proteins ◽

Human Growth ◽

Soluble Form ◽

Fusion Partner ◽

Small Protein ◽

E Coli ◽

Tev Protease

Abstract Background Growth factors (GFs) are signaling proteins that affect cellular processes such as growth, proliferation, and differentiation. GFs are used as cosmeceuticals, exerting anti-wrinkle, anti-aging, and whitening effects, and also as pharmaceuticals to treat wounds, growth failure, and oral mucositis. However, in mammalian and bacterial cells, low productivity and expression in inclusion bodies, respectively, of GFs does not satisfy the consumer demand. Here, we aimed to develop a bacterial expression system that produces high yields of soluble GFs that can be purified in their native forms. Results We present Fh8, an 8-kDa peptide from Fasciola hepatica with an N-terminal hexa-histidine (6HFh8), as a fusion partner for enhanced human GF production in recombinant Escherichia coli. The fusion partner harboring a tobacco etch virus (TEV) protease cleavage site was fused to the N-terminus of 10 human GFs: acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), epidermal growth factor (EGF), human growth hormone (hGH), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor 165 (VEGF165), keratinocyte growth factor 1 (KGF-1), placental growth factor (PGF), stem cell factor (SCF), and tissue inhibitor of metalloproteinase 1 (TIMP-1). The fusion proteins were expressed in E. coli under the control of T7 promoter at three temperatures (25 °C, 30 °C, and 37 °C). All individual fusion proteins, except for SCF and TIMP-1, were successfully overexpressed in cytoplasmic soluble form at more than one temperature. Further, the original aFGF, IGF-1, EGF, and VEGF165 proteins were cleaved from the fusion partner by TEV protease. Five-liter fed-batch fermentation approaches for the 6HFh8-aFGF (lacking disulfide bonds) and 6HFh8-VEGF165 (a cysteine-rich protein) were devised to obtain the target protein at concentrations of 9.7 g/l and 3.4 g/l, respectively. The two GFs were successfully highly purified (> 99% purity). Furthermore, they exerted similar cell proliferative effects as those of their commercial equivalents. Conclusions We demonstrated that 6HFh8-GF fusion proteins could be overexpressed on a g/l scale in the cytoplasm of E. coli, with the GFs subsequently highly purified and maintaining their biological activity. Hence, the small protein 6HFh8 can be used for efficient mass-production of various GFs.

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