TY - JOUR
T1 - Production of refolded Toxoplasma gondii recombinant SAG1-related sequence 3 (SRS3) and its use for serodiagnosis of human toxoplasmosis
AU - Mirzadeh, Abolfazl
AU - Saadatnia, Geita
AU - Golkar, Majid
AU - Babaie, Jalal
AU - Noordin, Rahmah
N1 - Funding Information:
Partial funding of this project was provided by the Ministry of Health and Medical Education, Iran and the Eastern Mediterranean Health Genomics and Biotechnology Network (EMGEN) fund provided under a grant through the Deputy of Research and Technology, Ministry of Health and Medical Education, Iran (grant no. 20134).
Publisher Copyright:
© 2017 Elsevier Inc.
PY - 2017/5/1
Y1 - 2017/5/1
N2 - SAG1-related sequence 3 (SRS3) is one of the major Toxoplasma gondii tachyzoite surface antigens and has been shown to be potentially useful for the detection of toxoplasmosis. This protein is highly conformational due to the presence of six disulfide bonds. To achieve solubility and antigenicity, SRS3 depends on proper disulfide bond formation. The aim of this study was to over-express the SRS3 protein with correct folding for use in serodiagnosis of the disease. To achieve this, a truncated SRS3 fusion protein (rtSRS3) was produced, containing six histidyl residues at both terminals and purified by immobilized metal affinity chromatography. The refolding process was performed through three methods, namely dialysis in the presence of chemical additives along with reduced/oxidized glutathione and drop-wise dilution methods with reduced/oxidized glutathione or reduced DTT/oxidized glutathione. Ellman's assay and ELISA showed that the protein folding obtained by the dialysis method was the most favorable, probably due to the correct folding. Subsequently, serum samples from individuals with chronic infection (n = 76), probable acute infection (n = 14), and healthy controls (n = 81) were used to determine the usefulness of the refolded rtSRS3 for Toxoplasma serodiagnosis. The results of the developed IgG-ELISA showed a diagnostic specificity of 91% and a sensitivity of 82.89% and 100% for chronic and acute serum samples, respectively. In conclusion, correctly folded rtSRS3 has the potential to be used as a soluble antigen for the detection of human toxoplasmosis.
AB - SAG1-related sequence 3 (SRS3) is one of the major Toxoplasma gondii tachyzoite surface antigens and has been shown to be potentially useful for the detection of toxoplasmosis. This protein is highly conformational due to the presence of six disulfide bonds. To achieve solubility and antigenicity, SRS3 depends on proper disulfide bond formation. The aim of this study was to over-express the SRS3 protein with correct folding for use in serodiagnosis of the disease. To achieve this, a truncated SRS3 fusion protein (rtSRS3) was produced, containing six histidyl residues at both terminals and purified by immobilized metal affinity chromatography. The refolding process was performed through three methods, namely dialysis in the presence of chemical additives along with reduced/oxidized glutathione and drop-wise dilution methods with reduced/oxidized glutathione or reduced DTT/oxidized glutathione. Ellman's assay and ELISA showed that the protein folding obtained by the dialysis method was the most favorable, probably due to the correct folding. Subsequently, serum samples from individuals with chronic infection (n = 76), probable acute infection (n = 14), and healthy controls (n = 81) were used to determine the usefulness of the refolded rtSRS3 for Toxoplasma serodiagnosis. The results of the developed IgG-ELISA showed a diagnostic specificity of 91% and a sensitivity of 82.89% and 100% for chronic and acute serum samples, respectively. In conclusion, correctly folded rtSRS3 has the potential to be used as a soluble antigen for the detection of human toxoplasmosis.
KW - In vitro refolding
KW - Recombinant
KW - SAG1-Related sequence 3(SRS3)
KW - Serodiagnosis
KW - Toxoplasma gondii
UR - http://www.scopus.com/inward/record.url?scp=85014881064&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2017.03.001
DO - 10.1016/j.pep.2017.03.001
M3 - Article
C2 - 28263855
AN - SCOPUS:85014881064
SN - 1046-5928
VL - 133
SP - 66
EP - 74
JO - Protein Expression and Purification
JF - Protein Expression and Purification
ER -