Characterisation of a Teladorsagia circumcincta glutathione transferase

Abstract

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

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

“…Recombinant TciALDO-1 was produced as recombinant poly-histidine protein, and was obtained by FPLC under native conditions using a Ni-NTA column (Qiagen, Germantown, MD, USA), and a Biologic DUO-FLOW BIO-RAD chromatography system (Bio-Rad, Auckland, New Zealand) as described before [24]. The protein was eluted using 500 mM imidazole, dialyzed overnight, and the concentration determined using the A280 nm assay with extinction coefficient (34,755 M −1 cm −1 ) and molecular weight (43.8 KDa).…”

“…The structural model of TciALDO-1 was generated from the amino acid sequence as previously described by [24,25]. Due to the lack of a perfect homologues template in PDB, homology modelling of the TciALDO-1 protein sequence was not possible, and therefore we used the threading method to model the protein structure using I-TASSER server [26].…”

“…The structural model of TciALDO-1 was generated from the amino acid seq previously described by [24,25]. Due to the lack of a perfect homologues templat homology modelling of the TciALDO-1 protein sequence was not possible, and we used the threading method to model the protein structure using I-TASSER se The selected model had a C-score of −0.11, and a TM value of 0.70 ± 0.12, where score represents a metric of the degree of similarity of the two protein structure model was based on [27].…”

Characterisation and Recognition by Immune Hosts of a Sheep Nematode Parasite Teladorsagia circumcincta Chitinase

“…E. coli strain BL21 (DE3) transformed with AY2.4 TciCHT as described previously [21] was grown in Luria Broth (LB). L-Arabinose was added as inducer and the culture grown for an additional 2h at 30 oC and 250rpm.…”

Multi-omic Profiling, Structural Characterization and Potent Inhibitor Screening of Evasion Related Proteins of a Parasitic Nematode, Haemonchus contortus, Surviving Vaccine Treatment

“…A vaccine (Barbervax®) based on an extract of adult H. contortus has recently been commercialized in Australia [5]. Our recent work aimed aimed at developing a recombinant Haemonchus vaccine comprising of 11 antigens has proven effective in weaned lambs as well as the closely related Teladorsagia circumcincta vaccine [6][7][8][9], however further details around the vaccine composition and efficacy cannot be released.…”