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

2019 ◽  
Vol 10 (4) ◽  
pp. 3319-3324 ◽  
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.

scholarly journals Purification and parcial characterization of a hemagglutinating factor (HAF): a possible adhesive factor of the diffuse adherent of Escherichia coli (DAEC)

1996 ◽  
Vol 38 (6) ◽  
pp. 401-406 ◽  
Author(s):  
Yano Tomomasa ◽  
Cleide Ferreira Catani ◽  
Michiko Arita ◽  
Takeshi Honda ◽  
Toshio Miwatani
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Hela Cells ◽  
Immunofluorescence Test ◽  
Native Form ◽  
Bacterial Surface ◽  
E Coli ◽  
Sds Page ◽  
Adhesive Factor

The mannose-resistant hemagglutinating factor (HAF) was extracted and purified from a diffuse adherent Escherichia coli (DAEC) strain belonging to the classic enteropathogenic E. coli (EPEC) serotype (0128). The molecular weight of HAF was estimated to be 18 KDa by SDS-PAGE and 66 KDa by Sephadex G100, suggesting that the native form of HAF consists of 3-4 monomeric HAF. Gold immunolabeling with specific HAF antiserum revealed that the HAF is not a rigid structure like fimbriae on the bacterial surface. The immunofluorescence test using purified HAF on HeLa cells, in addition to the fact that the HAF is distributed among serotypes of EPEC, suggests that HAF is a possible adhesive factor of DAEC strains

scholarly journals Expression of Lon-like Protease Gene from Lactobacillus plantarum IIA-1A5 in Escherichia coli BL21(DE3)

2019 ◽  
Vol 19 (2) ◽  
pp. 149-158
Author(s):  
Olfa Mega ◽  
Cece Sumantri ◽  
Irma Isnafia Arief ◽  
Cahyo Budiman
Keyword(s):  
Molecular Weight ◽  
Lactobacillus Plantarum ◽  
Expression System ◽  
Gc Content ◽  
Whole Genome Sequence ◽  
Protease Gene ◽  
Iptg Induction ◽  
E Coli ◽  
Sds Page ◽  
Index Value

Proteases are one of most important and abundant enzymes produced by the biotechnology industry, for scientific, physiological and industrial application and dominates of the whole enzyme market. Lactobacillus plantarum IIA-1A5 is an Indonesian lactic acid bacteria (LAB) isolated from beef Peranakan Ongole cattle. Preliminary analysis on its whole genome sequence indicated that this strain harbours some genes involved in protein degradation and might be promising to be further applied. This study aims to optimize the gene sequence of a lon-like protease of L. plantarum IIA-1A5 for heterologous expression system. The Lon-like gene expression system is made using genes that have been optimized first in silico.  pET-28a(+), E. coli BL21(DE3), Nde1 and BamH1 were used in this study as a expression vector, a host and retriction enzyme, respectively.  Molecular weight was validated using SDS-PAGE and expasy.org software. The results showed that optimization increased codon adaptation index value (CAI) and GC content to 0.97 and 56.57%, respectively, which were suitable for the E. coli expression system. The Lon-like IIA gene was successfully expressed in the cell cytoplasm by induction of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C.  As many as 88% of Lon-like IIA codons were distributed in the 91-100 quality group. Lon-like IIA was successfully expressed in a host cell induced with 1 mM IPTG at 37oC . IPTG induction was performed at the 3rd hour of incubation with OD600 0.59. In addition, Lon-like IIA molecular weight was detected approximately 43 kDa.

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

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Garshasb Rigi ◽  
Amin Rostami ◽  
Habib Ghomi ◽  
Gholamreza Ahmadian ◽  
Vasiqe Sadat Mirbagheri ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Hormone ◽  
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 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.

scholarly journals EXPRESSION OF CELLULOSE-DEGRADING ENDOGLUCANASE FROM BACILLUS SUBTILIS USING PTOLT EXPRESSION SYSTEM IN ESCHERICHIA COLI

2021 ◽  
Vol 55 (5-6) ◽  
pp. 619-627
Author(s):  
HÜLYA KUDUĞ CEYLAN ◽  
YAKUP ULUSU ◽  
SEMA BILGIN ◽  
İSA GÖKÇE
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Expression System ◽  
Industrial Applications ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
E Coli ◽  
Glycosidic Bonds ◽  
Sds Page ◽  
Dna Fragment

Endoglucanases randomly hydrolyse the cellulose chains by acting upon internal β-1,4-D-glycosidic bonds and are used extensively in industrial applications. In this study, bacterial endoglucanase gene yhfE was obtained by PCR, using primers based on genomic sequences of Bacillus subtilis strains. 1041 bp DNA fragment of yhfE was cloned into Escherichia coli DH5α through the use of pTolT expression plasmid. PCR, restriction enzyme analysis and DNA sequencing were performed in order to confirm the cloning. E. coli BL21-AI cells expressed the yhfE after induction at 0.04% of arabinose concentration for 4 h. The expected 38.7 kDa size yhfE protein after digestion with thrombin of the His-tagged fusion protein (yhfE-TolAIII) was visualized by SDS-PAGE. The yhfE-TolAIII production yield was approximately 82 mg/L. The recombinant yhfE was characterized by MALDI-TOF mass spectrometry and CD analysis.

scholarly journals An assay of human tyrosine protein kinase ABL activity using an Escherichia coli protein expression system

BioTechniques ◽  
2021 ◽  
Author(s):  
Emiko Kinoshita-Kikuta ◽  
Momoka Yoshimoto ◽  
Marina Yano ◽  
Eiji Kinoshita ◽  
Tohru Koike
Keyword(s):  
Escherichia Coli ◽  
Protein Kinase ◽  
Expression System ◽  
Kinase Domain ◽  
Wild Type ◽  
E Coli ◽  
Abl Kinase ◽  
Sds Page ◽  
Tyrosine Protein Kinase ◽  
Comparative Results

ABL, a human tyrosine protein kinase, and its substrate are co-expressed in Escherichia coli. Tyrosine phosphorylation of the substrate in E. coli was detected using Phos-tag SDS-PAGE. The bacterial co-expression system was used as a field for the kinase reaction to evaluate the enzymatic activity of five types of ABL kinase domain mutants. Relative to wild-type ABL, kinase activity was comparable in the H396P mutant, reduced in both Y253F and E255K mutants and undetectable in T315I and M351T mutants. These comparative results demonstrated that the phosphorylation states of the mutants correlated with their activity. The bacterial co-expression system permits rapid production of tyrosine kinase variants and provides a simple approach for examining their structure–activity relationships.

Alterations in lipopolysaccharide produced by chemostat-grown Escherichia coli O157:H7 as a function of growth rate and growth-limiting nutrient

1987 ◽  
Vol 33 (5) ◽  
pp. 452-458 ◽  
Author(s):  
Karen L. Dodds ◽  
Malcolm B. Perry ◽  
Ian J. McDonald
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Growth Rate ◽  
High Molecular Weight ◽  
Growth Rates ◽  
Sodium Dodecyl ◽  
High Growth ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Sds Page

Escherichia coli O157:H7 was grown in chemostats as continuous cultures at different controlled growth rates and under different nutrient limitations to determine the effects on lipopolysaccharide (LPS) structure. LPS from whole cells and extracted using the hot aqueous phenol method was examined by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and by gel filtration after hydrolysis with acetic acid. At low growth rates under glucose limitation (D = 0.1 h−1, doubling time (td), approx. 416 min; or D = 0.4 h−1, td, approx. 104 min), E. coli O157 produced high molecular weight LPS identical to that previously characterized from cells grown in batch culture. At a high growth rate (D = 0.8 h−1, td, approx. 52 min), the ratio of high molecular weight LPS to low molecular weight LPS produced greatly decreased. A small amount of high molecular weight LPS, containing O-polysaccharide which lacked amino sugars, and which thus was chemically different from that previously characterized, was produced by the cells at high growth rates. The predominant form of LPS from these cells was of slightly higher molecular weight than rough LPS, probably S–R LPS, and it consistently formed aggregates on SDS–PAGE. This form of LPS was also predominant when E. coli O157 was grown under Mg2+ limitation at an intermediate growth rate (D = 0.4 h−1, td, approx. 104 min).

scholarly journals Design, Expression and Purification of Strongyloides stercoralis IgG4 Immunoreactive Protein (NIE) in Escherichia coli

2020 ◽  
Author(s):  
Katayoun DASTAN ◽  
Mehdi ASSMAR ◽  
Nour AMIRMOZAFARI ◽  
Fariborz Mansour GHANAEI ◽  
Mirsasan MIRPOUR
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Western Blotting ◽  
Expression System ◽  
Strongyloides Stercoralis ◽  
Health Concern ◽  
Immunoreactive Protein ◽  
E Coli ◽  
Elisa Kit ◽  
Sds Page

Background: Strongyloidiasis is a public health concern in northern regions of Iran, caused by Strongyloides stercoralis. Auto-infection cycle can be resulted in high parasitic load, especially in immunocompromised hosts. Because of low sensitivity of stool culture and stool-based microscopy techniques, detection of antibodies in patient’s sera can be an alternative diagnostic technique for detection of the nematode. In the present study, as the first step of the development of an ELISA kit for the detection of antibodies against the nematode, IgG4 immunoreactive protein (NIE) was expressed in Escherichia coli expression system, purified and verified. Methods: The NIE gene sequence was retrieved from the GenBank. This sequence was codon-optimized for the expression in E. coli BL21 (DE3). The sequence was inserted into the expression vector pET-30b (+). The recombinant vector was then transferred into competent E. coli BL21 (DE3). Transformed colonies were selected and verified by colony PCR. NIE gene expression was induced with IPTG induction. The protein production was evaluated by SDS-PAGE and verified using Western blotting. Results: The codon-optimized NIE gene had required parameters for expression in E. coli. NIE protein was proved and verified by SDS-PAGE and Western blotting.  Conclusion: NIE recombinant protein was successfully expressed in E. coli expression system in appropriate amounts. The recombinant protein can be used for developing ELISA kit in diagnosis of S. stercoralis.

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

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.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

scholarly journals Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 691-702 ◽  
Author(s):  
B L Berg ◽  
V Stewart
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Formate Dehydrogenase ◽  
Recombinant Dna ◽  
Structural Genes ◽  
Respiratory Pathway ◽  
E Coli ◽  
Formate Oxidation ◽  
Operon Expression ◽  
K 12

Abstract Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the fdn operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

Sign in / Sign up

Export Citation Format

Share Document