Characterisation of a Teladorsagia circumcincta glutathione transferase

ABSTRACTA 615 bp full length cDNA encoding a Teladorsagia circumcincta glutathione transferase (TcGST) was cloned, expressed in Escherichia coli and the recombinant protein purified and its kinetic properties determined. The predicted protein consisted of 205 amino acids and was present as a single band of about 24 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TcGST with homologues from other helminths showed that the highest identity of 53-68% with haem-binding nematode proteins designated as members of the nu class of GSTs. Substrate binding sites and conserved regions were identified and were generally conserved. The predicted 3-dimensional structures of TcGST and HcGST revealed highly open binding cavities typical of this class of GST, considered to allow greater accessibility to diverse ligands compared with other classes of GST. At 25 °C, the optimum pH for TcGST activity was pH 7, the Vmax was 1535 ± 33 nmoles.min-1.mg-1 protein and the apparent Km for the substrate 1-chloro-2,4-dinitrobenzene (CDNB) was 0.22 ± 0.01 mM (mean ± SD, n = 2). Antibodies in both serum and saliva from field-immune, but not nematode-naÏve, sheep, recognised recombinant TcGST in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins. These findings could aid in the design of novel drugs and vaccine antigens for economically important parasites of livestock.

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Teladorsagia circumcincta 1,6 bisphosphate aldolase: molecular and biochemical characterisation, structure analysis and recognition by immune hosts

10.1101/2020.07.13.201335 ◽

2020 ◽

Author(s):

S. Umair ◽

C.L.G. Bouchet ◽

N. Palevich ◽

J.S. Knight ◽

H.V. Simpson

Keyword(s):

Recombinant Protein ◽

Kinetic Properties ◽

Dictyocaulus Viviparus ◽

Teladorsagia Circumcincta ◽

Multiple Alignments ◽

Sds Page ◽

Biochemical Characterisation ◽

Optimum Ph ◽

Fructose 1,6 Bisphosphate ◽

Substrate Binding Sites

ABSTRACTA 1095 bp full length cDNA encoding Teladorsagia circumcincta aldolase (TciALDO) was cloned, expressed in Escherichia coli, the recombinant protein purified and its kinetic properties determined. A phylogenetic tree was constructed using helminth aldolase sequences. The predicted protein consisted of 365 amino acids and was present as a single band of about 44 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TciALDO with homologues from other helminths showed that the greatest similarity (93%) to the aldolases of Haemonchus contortus and Dictyocaulus viviparus, 82-86% similarity to the other nematode sequences and 68-71% similarity to cestode and trematode enzymes. Substrate binding sites and conserved regions were identified and were completely conserved in other homologues. At 25 °C, the optimum pH for TciALDO activity was pH 7.5, the Vmax was 432 ± 23 nmoles.min−1.mg−1 protein and the apparent Km for the substrate fructose 1,6-bisphosphate was 0.24 ± 0.01 μM (mean ± SEM, n = 3). Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TciALDO in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to native aldolase indicates similar antigenicity of the two proteins.

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Teladorsagia circumcincta 1,6-Bisphosphate Aldolase: Molecular and Biochemical Characterisation, Structure Analysis and Recognition by Immune Hosts

Parasitologia ◽

10.3390/parasitologia1010001 ◽

2021 ◽

Vol 1 (1) ◽

pp. 1-11

Author(s):

Saleh Umair ◽

Charlotte Bouchet ◽

Nikola Palevich ◽

Heather Simpson

Keyword(s):

Binding Sites ◽

Kinetic Properties ◽

Dictyocaulus Viviparus ◽

Teladorsagia Circumcincta ◽

Multiple Alignments ◽

Sds Page ◽

Biochemical Characterisation ◽

Optimum Ph ◽

Fructose 1,6 Bisphosphate ◽

Substrate Binding Sites

A 1095 bp full length cDNA encoding Teladorsagia circumcincta aldolase (TciALDO-1) was cloned and expressed in Escherichia coli. Recombinant TciALDO-1 was purified, and its kinetic properties determined. The predicted protein consisted of 365 amino acids, and was present as a single band of about 44 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TciALDO-1 with homologues from other helminths showed the greatest similarity (93%) to the aldolases of Haemonchus contortus and Dictyocaulus viviparus, 82–86% similarity to the other nematode sequences, and 68–71% similarity to cestode and trematode enzymes. Substrate binding sites and conserved regions were identified, and were completely conserved in other homologues. At 30 °C, the optimum pH for TciALDO-1 activity was pH 7.5, the Vmax was 432 ± 23 nmol × min−1 × mg−1 protein, and the apparent Km for the substrate fructose 1,6-bisphosphate was 0.24 ± 0.01 µM (mean ± SEM, n = 3). Recombinant TciALDO-1 was recognized by antibodies in both serum and saliva from field-immune sheep in ELISA, however, that was not the case with nematode-naïve sheep. Teladorsagia circumcincta fructose 1,6-bisphosphate aldolase appears to have potential as a vaccine candidate to control this common sheep parasite.

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Structural features, properties and regulation of the outer-membrane protein W (OmpW) of Vibrio cholerae

Microbiology ◽

10.1099/mic.0.27995-0 ◽

2005 ◽

Vol 151 (9) ◽

pp. 2975-2986 ◽

Cited By ~ 65

Author(s):

Bisweswar Nandi ◽

Ranjan K. Nandy ◽

Amit Sarkar ◽

Asoke C. Ghose

Keyword(s):

Membrane Protein ◽

Recombinant Protein ◽

Outer Membrane ◽

Vibrio Cholerae ◽

Outer Membrane Protein ◽

Secondary Structure Prediction ◽

Sequence Data ◽

Insertion Mutagenesis ◽

Sds Page

The outer-membrane protein OmpW of Vibrio cholerae was studied with respect to its structure, functional properties and regulation of expression. On SDS-PAGE, the membrane-associated form of OmpW protein (solubilized by either 0·1 % or 2 % SDS at 25 °C) migrated as a monomer of 19 kDa that changed to 21 kDa on boiling. The protein was hyperexpressed in Escherichia coli in the histidine-tagged form and the purified His6-OmpW (heated or unheated) migrated as a 23 kDa protein on SDS-PAGE. Circular dichroism and Fourier-transform infrared spectroscopic analyses of the recombinant protein showed the presence of β-structures (∼40 %) with minor amounts (8–15 %) of α-helix. These results were consistent with those obtained by computational analysis of the sequence data of the protein using the secondary structure prediction program Jnet. The recombinant protein did not exhibit any porin-like property in a liposome-swelling assay. An antiserum to the purified protein induced a moderate level (66·6 % and 33·3 % at 1 : 50 and 1 : 100 dilutions, respectively) of passive protection against live vibrio challenge in a suckling mouse model. OmpW-deficient mutants of V. cholerae strains were generated by insertion mutagenesis. In a competitive assay in mice, the intestinal colonization activities of these mutants were found to be either only marginally diminished (for O1 strains) or 10-fold less (for an O139 strain) as compared to those of the corresponding wild-type strains. The OmpW protein was expressed in vivo as well as in vitro in liquid culture medium devoid of glucose. Interestingly, the glucose-dependent regulation of OmpW expression was less prominent in a ToxR− mutant of V. cholerae. Further, the expression of OmpW protein was found to be dependent on in vitro cultural conditions such as temperature, salinity, and availability of nutrients or oxygen. These results suggest that the modulation of OmpW expression by environmental factors may be linked to the adaptive response of the organism under stress conditions.

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Cloning, expression and immunogenic characterization of theapxIIAgene ofActinobacillus pleuropneumoniae

Chinese Journal of Agricultural Biotechnology ◽

10.1017/s1479236207001222 ◽

2007 ◽

Vol 4 (1) ◽

pp. 63-67

Author(s):

Yan Ke-Xia ◽

Liu Jian-Jie ◽

Wu Bin ◽

Tang Xi-Biao ◽

Cai Li-Jun ◽

...

Keyword(s):

Recombinant Protein ◽

Lethal Dose ◽

Serum Antibody ◽

Enzyme Linked Immunosorbent Assay ◽

Gene Encoding ◽

Lethal Challenge ◽

Sds Page ◽

Prokaryotic Expression Vector ◽

Protection Rate ◽

Protein Group

AbstractThe structural gene encoding ApxII toxin (apxIIA) was amplified from the genomic DNA ofActinobacillus pleuropneumoniae(APP) strain HB08 (serotype 2) and cloned into the prokaryotic expression vector pET-28a. SDS-PAGE and Western blot analysis showed that theapxIIAgene was expressed inEscherichia coliBL21 (DE3) and the expressed products could react with ApxII antibodies. The recombinant ApxIIA was purified from the inclusion bodies. Kunming mice were intraperitoneally vaccinated twice, with an interval of 2 weeks, using unfolded/refolded recombinant proteins, the native ApxII toxin extracted from the cultural supernatant of a strain of APP serotype 7 (APP-7) or phosphate-buffered saline (PBS). Serum antibody was examined by ApxIIA-specific enzyme-linked immunosorbent assay (ELISA) 2 weeks after every vaccination. Two weeks after the second vaccination, mice were challenged intraperitoneally with a lethal dose of APP-7 (1.08 × 108cfu per mouse). The protection rate reached 91.7% in the native ApxII group, 83.3% in the refolded recombinant protein group and 58.3% in the unfolded recombinant protein group, while all mice in the PBS group died within 36 h after challenge. Our data revealed that the refolded recombinant ApxIIA had excellent immunogenicity and could elicit protection against a lethal challenge of APP.

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Immobilization of Purified Fungal Laccase on Cost Effective Green Coconut Fiber and Study of its Physical and Kinetic Characteristics in Both Free and Immobilized Form

Current Biotechnology ◽

10.2174/2211550108666190201151816 ◽

2019 ◽

Vol 8 (1) ◽

pp. 3-14 ◽

Cited By ~ 1

Author(s):

Priyanka Ghosh ◽

Uma Ghosh

Keyword(s):

Activation Energy ◽

Gel Filtration ◽

Cost Effective ◽

Industrial Applications ◽

Kinetic Properties ◽

Coconut Fiber ◽

Immobilized Laccase ◽

Sds Page ◽

Glutaraldehyde Solution ◽

Laccase Enzyme

Background: Laccases are important enzymes that have numerous applications in different biotechnological sectors. Objective: The aim was to purify laccase from Aspergillus flavus PUF5, successfully immobilize it on coconut fiber and characterize different physical and kinetic properties under both free and immobilize conditions. Methods: Laccase from A. flavus PUF5 was purified using ammonium sulfate precipitation, followed by DEAE column chromatography and gel filtration using Sephadex G100. The molecular weight was determined through SDS-PAGE (12%). It was immobilized on pretreated coconut fiber through crosslinking by glutaraldehyde (4% v/v). Physical and kinetic parameters like optimum temperature, pH, thermostability, the effect of additives, activation energy, Km and Vmax for free and immobilized laccase were also analyzed. Recycling stability of the immobilized laccase was further determined. Results: The extracellular laccase (65 kDa) was purified up to homogeneity and was immobilized on acid-pretreated coconut fiber by 4% (v/v) glutaraldehyde solution at 30°C, pH 5.0. Activation energy (Ea) of free and immobilized laccase for oxidation of guaiacol was found to be 24.69 and 32.76 kJ mol-1 respectively. Immobilized laccase showed higher melting temperature (Tm) of (82.5°C) than free enzyme (73°C). Km and Vmax for free and immobilized laccase were found to be 0.67 mM, 0.70 mM and 280 U/mg, 336 U/mg respectively when guaiacol was used as substrate. Additionally, in immobilized condition laccase retained ˃80% of its initial activity after use till six repeated cycles. Conclusion: The purified laccase enzyme and the cheap immobilization seem to be a prospective process for different biotechnological and industrial applications.

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Expression, Purification and Preparation of Polyclonal Antibody of Bombyx mori Serpin-6

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.796.72 ◽

2013 ◽

Vol 796 ◽

pp. 72-76

Author(s):

Ming Hui Wang ◽

Kai-Zun Xu ◽

Guo Sheng Li ◽

Yan Yun Wang ◽

Wei De Shen ◽

...

Keyword(s):

Recombinant Protein ◽

Protein Expression ◽

Protease Inhibitor ◽

Bombyx Mori ◽

Serine Protease ◽

Polyclonal Antibody ◽

Antibody Test ◽

Serine Protease Inhibitor ◽

Sds Page ◽

Practical Tool

Serpin (Serine Protease Inhibitor) widely distributes in animals, plants, protozoan, prokaryotes and viruses. Serpin-6 belongs to the superfamily of serine protease inhibitor. In this study, we constructed a prokaryotic vector - pET28a-serpin-6, and used IPTG (isopropyl thiogalactoside) to induce serpin-6 protein expression, then applied the Ni-affinity chromatography to purify the collected recombinant protein. The assay of SDS-PAGE and Anti-his Polyclonal antibody Test showed that the recombinant serpin-6 protein have a molecular weight of 45 kDa. The highly purified sample of the recombinant protein was obtained. The protein has been used as antigen to immunizeKunMing miceusing the 4 times immunization method. We successfully attained the polyclonal antibody of serpin-6.The titer of the antibody is as high as 1:20000, with a good specificity. The polyclonal antibody can provide a practical tool to further study the distribution of protein expression and functions of serpin-6 in different states and breeds ofBombyx mori.

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

Science and Technology Development Journal - Natural Sciences ◽

10.32508/stdjns.v1it1.436 ◽

2017 ◽

Vol 1 (T1) ◽

pp. 69-79 ◽

Cited By ~ 1

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.

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

Vietnam Journal of Biotechnology ◽

10.15625/1811-4989/18/2/14886 ◽

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.

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The Expression of The PfEMP1-DBL2β Recombinant Protein of Plasmodium falciparum Welch, 1897 Isolated From Indonesia

Jurnal ILMU DASAR ◽

10.19184/jid.v21i1.10494 ◽

2020 ◽

Vol 21 (1) ◽

pp. 67

Author(s):

Fathul Hidayatul Hasanah ◽

Erma Sulistyaningsih ◽

Widhi Dyah Sawitri

Keyword(s):

Plasmodium Falciparum ◽

Recombinant Protein ◽

Severe Malaria ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Restriction Enzymes ◽

Supernatant Fraction ◽

Intracellular Adhesion Molecule ◽

Pathological Mechanism ◽

Sds Page

The binding of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) to Intracellular Adhesion Molecule-1 (ICAM-1) is a major pathological mechanism in severe malaria including cerebral malaria. The binding is mediated by PfEMP1-DBL2β domain. The study aimed to explore there combinant protein of PfEMP1-DBL2β domain of P. falciparum isolated from Indonesia. DNA was isolated from a severe malaria patient. The DBL2β domain was amplified using Polymerase Chain Reaction (PCR) with specific primer and cloned into the pJET1 cloning vector. The DBL2β recombinant protein was constructed from DBL2β-pJET1 clone using pET-30a expression vector and expressed in Escherichia coli BL21-DE3. PCR colony and digestion of plasmid clones using restriction enzymes were conducted to confirm cloning result, and the expression of recombinant protein was analyzed using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The expression of DBL2β-PfEMP1 domain is higher in pellet than in supernatant fraction. In conclusion, the DBL2β-PfEMP1 domain recombinant protein of P. falciparum isolated from Indonesia expressed as a ~66 kDa protein in full length. Keywords: DBL2β domain, Indonesia, PfEMP1, Plasmodium falciparum, recombinant protein.

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

The Journal of Infection in Developing Countries ◽

10.3855/jidc.1557 ◽

2011 ◽

Vol 5 (12) ◽

pp. 856-862 ◽

Cited By ~ 7

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.

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