Mahmoudi, Elahe, Kalhor, Hamid Reza, Yadegari, Mohammad Hossein, Sadeghizadeh, Majid, Sadeghizadeh, Majid, Hassan, Zohair (2010). Expression, purification and investigation of sap2 Candida albican’s activity in pichia pasturis yeast system. , 12(4), 71-83.
Elahe Mahmoudi; Hamid Reza Kalhor; Mohammad Hossein Yadegari; Majid Sadeghizadeh; Majid Sadeghizadeh; Zohair Hassan. "Expression, purification and investigation of sap2 Candida albican’s activity in pichia pasturis yeast system". , 12, 4, 2010, 71-83.
Mahmoudi, Elahe, Kalhor, Hamid Reza, Yadegari, Mohammad Hossein, Sadeghizadeh, Majid, Sadeghizadeh, Majid, Hassan, Zohair (2010). 'Expression, purification and investigation of sap2 Candida albican’s activity in pichia pasturis yeast system', , 12(4), pp. 71-83.
Mahmoudi, Elahe, Kalhor, Hamid Reza, Yadegari, Mohammad Hossein, Sadeghizadeh, Majid, Sadeghizadeh, Majid, Hassan, Zohair Expression, purification and investigation of sap2 Candida albican’s activity in pichia pasturis yeast system. , 2010; 12(4): 71-83.

Expression, purification and investigation of sap2 Candida albican’s activity in pichia pasturis yeast system

Pathobiology Reserach
Article 8, Volume 12, Issue 4, 2010, Pages 71-83    PDF (352.43 K)
Authors
Elahe Mahmoudi1; Hamid Reza Kalhor2; Mohammad Hossein Yadegari* 3; Majid Sadeghizadeh4; Majid Sadeghizadeh4; Zohair Hassan5
1Ph.D. Student, Department of Medical Mycology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
2Assistant Professor, Department of Biochemistry, Faculty of Biolocical Sciences, Tarbiat Modares University, Tehran, Iran
3Assistant Professor, Department of Medical Mycology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
4Professor, Department of Genetics, Faculty of Biolocical Sciences, Tarbiat Modares University, Tehran, Iran
5Professor, Department of Medical Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Abstract
Objective: The secreted aspartic proteinases (Sap2) of Candida albicans has prominent role on Candida adherence, invasion, and pathogenicity. The aim of this study was cloning, expression and characterizing of Sap2 enzyme. Also in this study for the first time, the expression system P. pasturis was used for expressing the recombinant protein.
Materials and Methods: C. albicans Sap2 gene was amplified by PCR with sticky ends, EcoR1 and SacII, and it was subcloned into the T/A vector. The sequencing of this gene was done with universal primers and then the Sap2 gene was cloned into pGAPZαA expression vector. The construct was transformed into P. pasturis yeast; the Sap2 gene integration into the yeast genome was accomplished by the homologous recombination. The expressed protein was confirmed by western blotting using monoclonal antibody against Sap2 protein. Finally, the recombinant protein was purified by Ni-NTA chromatography column, and the activity of the enzyme was confirmed.
Results: In this study, we successfully amplified C.albicans Sap2 gene and subsequently integrated into the yeast pichia pasturis genome by homologous recombination. Moreover, we were able to identify a yeast clone secreting the recombinant protein. The optimum over expression of sap2 protein was obtained after 96 h, at 30ْ C.
Conclusion: Expression of Sap2 gene in P. pasturis, in comparison to bacterial expression system, leads to a high-level expression, and also need for post translation modifications, that might be required for the activity of enzyme, is obviated in the yeast system. Based on our results, the purified acid aspartyl proteinase purified from P. pasturis was capable of degrading BSA as a substrate in-vitro. The recombinant Sap2 protein had maximum activity in an acidic pH.
Keywords
C. albicans; Sap2 cloning; P. pasturis
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