scholarly journals Cloning and expression of maltooligosyltrehalose trehalohydrolase from Sulfolobus solfataricus DSM 1616 in Bacillus subtilis WB800

2020 ◽  
Vol 18 (2) ◽  
pp. 363-372
Author(s):  
Nguyen Tien Cuong ◽  
Nguyen Thi Hien Trang ◽  
Nguyen Thi Thao ◽  
Le Thanh Hoang ◽  
Nguyen Sy Le Thanh ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Protein ◽  
Amylase Activity ◽  
Expression System ◽  
Sulfolobus Solfataricus ◽  
Cloning And Expression ◽  
Exchange Event ◽  
Sds Page ◽  
Dna Fragment ◽  
Conventional Expression

Maltooligosyltrehalose trehalohydrolase (MTHase) is an industrial enzyme for the production of trehalose. A DNA fragment of 1680 bp encoding for MTHase was cloned from Sulfobolus solfataricus DSM 1616 then fused with promoter acoA-amyE already amplified from pMSE3 vector by PCR to generate an expression cassette acoMTH. Afterward the cassette was inserted into pAC7 vector for expression of the gene in Bacillus subtilis WB800 – a conventional expression system. Gene MTH was inserted into the genome of B. subtilis WB800 by cross-exchange event of pAC7 vector with the host genome for expression of high quality and high quantity of extracellular recombinant protein. By crossing-exchange event at 3’amyE-5’amyE, the expressional cassette was integrated into B. subtilis WB800 genome. The expressional cassette was integrated into B. subtilis WB800 genome replacing 3’amyE-5’amyE, hindering the native amylase activity of the host. Expression of expected protein was confirmed by electrophoresis SDS-PAGE. From our results, it indicates that gene MTH was expressed successfully in B. subtilis WB800. After 0.5% acetoin induction for 48 h, the data showed that the protein with a molecular mass of ~64 kDa on SDS-PAGE was expressed. The level of recombinant protein in WBpAacoMTH was increased and reached 2.5%, 15.2% and 21.95%, respectively comparing with native B. subtilis WB800.

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 Cloning and expression of haloacid dehalogenase gene from Bacillus cereus IndB1

2018 ◽  
Vol 22 (2) ◽  
pp. 55
Author(s):  
Enny Ratnaningsih ◽  
Idris Idris
Keyword(s):  
Recombinant Protein ◽  
Bacillus Cereus ◽  
Tertiary Structure ◽  
Specific Activity ◽  
Halogen Bond ◽  
Expression System ◽  
Industrial Sector ◽  
Cloning And Expression ◽  
Haloacid Dehalogenase ◽  
Organohalogen Compounds

Organohalogen compounds, widely used as pesticides in agriculture and solvents in the industrial sector, cause environmental pollution and health problems due to their toxicity and persistence. Numerous studies have been conducted on the biodegradation of organohalogen compounds, with many focusing on the use of dehalogenase from bacteria. Haloacid dehalogenase is a group of enzymes that cleaves the carbon-halogen bond in halogenated aliphatic acids. In a previous study, the bcfd1 gene encoded haloacid dehalogenase from Bacillus cereus IndB1 was successfully isolated and characterized. This research aimed to create an expression system of the bcfd1 gene by subcloning this gene into pET expression vector and to overexpress the gene in Escherichia coli BL21 (DE3). In addition, the recombinant protein was characterized to gain a better understanding of the catalytic action of this enzyme. A high expression of bcfd1 was obtained by inducing the culture at OD550 0.8–1.0  using 0.01 mM IPTG as determined by SDS-PAGE. Zymogram analysis proved that the recombinant protein possessed dehalogenase activity. Bcfd1 activity toward monochloroacetic acid (MCA) showed specific activity of 37 U/mg at 30°C, pH 9. The predicted tertiary structure of Bcfd1 was estimated has conserved α/ß hydrolase folding motif for haloacid dehalogenase superfamily.

scholarly journals Cloning and expression of a chick liver glutathione S-transferase CL 3 subunit with the use of a baculovirus expression system

1992 ◽  
Vol 281 (2) ◽  
pp. 545-551 ◽  
Author(s):  
L H Chang ◽  
J Y Fan ◽  
L F Liu ◽  
S P Tsai ◽  
M F Tam
Keyword(s):  
Specific Activity ◽  
Sequence Similarity ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Relative Activity ◽  
Cloning And Expression ◽  
Glutathione S Transferase ◽  
Sds Page ◽  
Glutathione S Transferases ◽  
A Subunit

Glutathione S-transferase CL 3 subunits purified from 1-day-old-chick livers were digested with Achromobacter proteinase I and the resulting fragments were isolated for amino acid sequence analysis. An oligonucleotide probe was constructed accordingly for cDNA library screening. A cDNA clone of 1342 bases, pGCL301, encoding a protein of 26209 Da was isolated and sequenced. Including conservative substitutions, this protein has 75-79% sequence similarity to other Alpha family glutathione S-transferases. The coding sequence of pGCL301 was inserted into a baculovirus vector for infection of Spodoptera frugiperda (SF9) cells. The expressed protein has a high relative activity with ethacrynic acid (47% of the specific activity with 1-chloro-2,4-dinitrobenzene). The enzyme has a subunit molecular mass of 25.2 +/- 1.2 kDa (by SDS/PAGE), a pI of 9.45 and an absorption coefficient A1%1cm of 13.0 +/- 0.5 at 280 nm.

scholarly journals Purification of p24 protein expressed in Bacillus subtilis and evaluation of its immunogenicity in mice

2017 ◽  
Vol 1 (T1) ◽  
pp. 69-79 ◽  
Author(s):  
Tuom Thi Tinh Truong ◽  
Trang Thi Phuong Phan ◽  
Hoang Duc Nguyen
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Protein ◽  
Western Blot ◽  
Host Cell ◽  
Essential Role ◽  
Subtilis Strain ◽  
Total Proteins ◽  
Over Expression ◽  
Sds Page ◽  
P24 Protein

p24 protein is a component of the HIV particle capsid. It plays an essential role in HIV to infect into the host cell and in the cycle life of virus. Therefore, this protein can be used in the orientative study “to create and produce HIV’s vaccine”. This study created the new Bacillus subtilis strain which expressed p24 protein. B. subtilis a safety and non-toxic bacteria strain for humans and animals, has system expression to allow over expression recombinant protein up to 10-30 % of total proteins. Plasmid pHT1537 was cloned successfully, containing lysSN-6his-gagp24 gene to encode p24 protein fused with LysSN protein and to allow the expression of p24 protein in B. subtilis by IPTG inducer. The target protein in the cell was cheked by SDS-PAGE. The p24 fused protein was parified from His Trap column which contained Ni2+. Evaluation of the ability to produce antibody against p24 protein in mice by ELISA and Western blot was caried out.

scholarly journals Cloning and Expression of Staphylococcus Simulans Lysostaphin Enzyme Gene in Bacillus Subtilis WB600

2020 ◽  
Author(s):  
Babak Elyasi Far ◽  
Mehran Ragheb ◽  
Reza Rahbar ◽  
Ladan Mafakher ◽  
Neda Yousefi Nojookambari ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Enzyme Stability ◽  
Expression System ◽  
Residual Activity ◽  
Maximum Activity ◽  
Cloning And Expression ◽  
Gene Fragment ◽  
Alkaline Lysis ◽  
Enzyme Gene ◽  
Staphylococcus Simulans

Abstract Background: Lysostaphin is a glycylglycine endopeptidase, secreted by Staphylococcus simulans, capable of specifically hydrolyzing pentaglycine crosslinks present in the peptidoglycan of the Staphylococcus aureus cell wall. In this paper, we describe the cloning and expression of the lysostaphin enzyme gene in Bacillus subtilis WB600 host using pWB980 expression system.Results: Plasmid pACK1 of S. simulans was extracted using alkaline lysis method. Lysostaphin gene was isolated by PCR and cloned into pTZ57R/T-Vector, then transformed into Escherichia coli DH5α. The amplified gene fragment and uncloned pWB980 vector were digested using PstI and XbaІ enzymes and purified. The restricted gene fragment was ligated into the pWB980 expression vector by the standard protocols, then the recombinant plasmid was transformed into B. subtilis WB600 using electroporation method. The recombinant protein was evaluated by the SDS-PAGE method and confirmed by western immunoblot. Analysis of the target protein showed a band corresponding to an about 27-kDa r-lysostaphin. Protein content was estimated 91µg/ml by Bradford assay.Conclusions: The recombinant lysostaphin represented 90% of its maximum activity at 40℃ and displayed good thermostability by keeping about 80% of its maximum activity at 45℃. Heat residual activity assay of recombinant lysostaphin demonstrated that the enzyme stability was up to 40℃ and showed good stability at 40℃ for 16 h incubation.

scholarly journals Cloning, expression and purification of outer membrane protein PorA of Neisseria meningitidis serogroup B

2011 ◽  
Vol 5 (12) ◽  
pp. 856-862 ◽  
Author(s):  
Fakhri Haghi ◽  
Shahin Najar Peerayeh ◽  
Seyed Davar Siadat ◽  
Mehran Montajabiniat
Keyword(s):  
Recombinant Protein ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Prokaryotic Expression ◽  
Membrane Vesicle ◽  
Outer Membrane Proteins ◽  
Expression System ◽  
Sds Page ◽  
Prokaryotic Expression System ◽  
Prokaryotic Expression Vector

Introduction: Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N. meningitidis serogroup B. PorA is a major component of the outer membrane of N. meningitidis and functions as a cationic porin. This study aimed to clone and determine the expression of PorA. Methodology: A 1200 bp fragment of porA gene was amplified by PCR from serogroup B N. meningitidis and then cloned into prokaryotic expression vector pET-32a. For expression of recombinant protein, pET32a-porA plasmid was transformed into competent Origami B (DE3) cells. Recombinant protein was overexpressed with isopropythio-beta-D-galctoside (IPTG) and affinity purified by Ni-NTA agarose. SDS-PAGE and western blotting were performed for protein determination and verification. Results: Cloning of porA was confirmed by colony-PCR and enzymatic digestion. In comparison with the corresponding sequences of original genes, the nucleotide sequence homology of the cloned porA gene was 97%. IPTG with a dosage of 1.0 mmol/L could efficiently induce protein expression. SDS-PAGE analysis showed that our constructed prokaryotic expression system pET32a-PorA-Origami efficiently produces a target recombinant protein with a molecular weight of 65 kDa. The recombinant PorA was overexpressed as inclusion bodies and reacted with the serum from a rabbit previously immunized with native outer membrane vesicle. Conclusion: This prokaryotic expression system provides an easy method for producing recombinant PorA and may also be useful for the production of other bacterial outer membrane proteins for vaccine studies.

scholarly journals Cloning and expression of LTB in Escherichia coli and Bacillus subtilis

2013 ◽  
Vol 16 (1) ◽  
pp. 13-22 ◽  
Author(s):  
Trang Thi Phuong Phan ◽  
Anh Le Tuan Nguyen ◽  
Hoang Duc Nguyen
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Fusion Protein ◽  
Expression System ◽  
Pharmaceutical Products ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
E Coli ◽  
B Subunit ◽  
The Common

LTB is the B subunit of heat labile toxins (LT) in Escherichia coli ETEC. This subunit is non-toxic but has a high immune response. Therefore, LTB is considered a suitable antigen for partial vaccine against the diarrhea caused by E. coli ETEC. The most important component of partial vaccine is antigen protein. Nowadays, with the advancement of recombinant protein technology, these antigens are mainly produced by the common bacterial expression system as E. coli. However, the recombinant proteins produced by E. coli are often miscellaneous with enterotoxins, which should be removed from pharmaceutical products. Thus, the production of antigen proteins in other expression system without endotoxins like Bacillus subtilis is in attention. We conducted the experiments of cloning and expressing LTB using a novel pHT plasmid that allow the protein to be expressed in both of E. coli and B. subtilis. We were successful to generate plasmid pHT326 and express the gene encoding for the fusion protein of LTB and LysSN-6xHis-TEV in B. subtilis and E. coli. The binding of fusion protein on the columns that have affinity with His-tag was confirmed. This result is about to be applied for the development of partial vaccine aganst the diarrhea as well as the development of some diagnostic kits for ETEC in food or medical waste and kits to detect antibodies against LTB in animals.

scholarly journals Possible Function of the ribT Gene of Bacillus subtilis: Theoretical Prediction, Cloning, and Expression

Acta Naturae ◽  
2014 ◽  
Vol 6 (3) ◽  
pp. 106-109 ◽  
Author(s):  
A. P. Yakimov ◽  
T. A. Seregina ◽  
A. A. Kholodnyak ◽  
R. A. Kreneva ◽  
A. S. Mironov ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Tertiary Structure ◽  
Protein Product ◽  
Cloning And Expression ◽  
Riboflavin Biosynthesis ◽  
E Coli ◽  
Sds Page ◽  
Phage T7 ◽  
Rna Polymerase Gene ◽  
Search For Homologs

The complete decipherment of the functions and interactions of the elements of the riboflavin biosynthesis operon (rib operon) of Bacillus subtilis are necessary for the development of superproducers of this important vitamin. The function of its terminal ribT gene has not been established to date. In this work, a search for homologs of the hypothetical amino acid sequence of the gene product through databases, as well as an analysis of the homolgs, was performed; the distribution of secondary structure elements was theoretically predicted; and the tertiary structure of the RibT protein was proposed. The ribT gene nucleotide sequence was amplified and cloned into the standard high-copy expression vector pET15b and then expressed after induction with IPTG in E. coli BL21 (DE3) strain cells containing the inducible phage T7 RNA polymerase gene. The ribT gene expression was confirmed by SDS-PAGE. The protein product of the expression was purified by affinity chromatography. Therefore, the real possibility of RibT protein production in quantities sufficient for further investigation of its structure and functional activity was demonstrated.

scholarly journals A novel multi-epitope recombinant protein for diagnosis of African swine fever

2020 ◽  
Author(s):  
ZHAN GAO ◽  
JUNJUN SHAO ◽  
GUANGLEI ZHANG ◽  
SUDAN GE ◽  
YANYAN CHANG ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
African Swine Fever Virus ◽  
Expression System ◽  
African Swine Fever ◽  
Economic Losses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Sds Page ◽  
Prokaryotic Expression System ◽  
Swine Diseases

Abstract Background: African swine fever(ASF) is an acute, severe and highly fatal infectious disease of pigs. The disease spreads rapidly, causing huge economic losses to the pig industry in infected areas. The structural proteins p30 and p54 in African swine fever virus(ASFV) have been verified as diagnostic antigens.Methods: In this study, we constructed a novel multi-epitope fusion antigen gene based on P30 and P54 proteins, induced expression in a prokaryotic expression system and analyzed the reactivity of the recombinant fusion protein. The purified recombinant protein m35 was used as the coating antigen to establish an indirect enzyme-linked immunosorbent assay (ELISA) detection method for ASFV. 116 serum samples and positive sera of other swine diseases were detected by indirect ELISA.Results: Our results indicate that the m35 gene fragment with a length of 558bp was successfully constructed. SDS-PAGE and Western Blotting analysis showed that the protein had a band at 22kDa, proving its good reactogenicity. ROC analysis was performed to validate the assay, the area under the ROC curve is 0.9738 (95% confidence interval, 0.9336 to 1.014), and does not cross-react with other swine diseases.Conclusion: Our results show that its sensitivity and specificity were highly accurate. It is feasible to use this recombinant protein as a diagnostic antigen to distinguish ASFV infection.

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.

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