Cloning, expression and purification of Leishmania major PSA-sfGFP fusion protein

Jordan Journal of Biological Sciences

article

Aisha Al-jaghasi¹  Abdul-Qader Abbady²  Sahar Al-Khatib¹  Chadi Soukkarieh¹ 

[1] Dept. of Animal Biology, Faculty of Sciences, Damascus University;Dept. of Molecular Biology and Biotechnology;Faculty of Pharmacy, Syrian Private University

关键词: PSA;    sfGFP;    Gene cloning;    Protein expression;   

DOI  :  10.54319/jjbs/140224

学科分类：生物科学（综合）

来源: Hashemite University, Deanship of Research and Higher Studies

【 摘 要 】

PSA (Promastigote Surface Antigen) is one of the immunogenic antigens in L. major. Protection depends on the source of PSA antigen, whereas native protein and plasmid DNA encoding PSA, but not recombinant PSA purified from E. coli provided significant protection. The green fluorescent protein (GFP) is commonly used as an excellent expression tag for fusion proteins, which can improve their expression while retaining their function and native-like structure. Trying to develop anti cutaneous leishmaniasis vaccine based on PSA (as recombinant protein or DNA vaccine), this study evaluated the cloning, expression, of the secreted PSA protein from L. major as a fusion partner to the superfolder form of green fluorescent protein (sfGFP) in both E. coli and HEK-293T cells. This included constructing protein expression plasmids pRSET-sfGFP-PSA and pcDNA-sfGFP-PSA, then producing the recombinant of 6× His tagged sfGFP-PSA protein (65 kDa) that was confirmed by SDS-PAGE and western blotting. Although sfGFP-PSA fusion protein was expressed with full length in both types of cells, a partial separation of sfGFP protein was observed when the fusion protein was expressed in E. coli. Here we presented an efficient method to produce the full length of PSA as a fusion protein with sfGFP, which could greatly facilitate its uses as a vaccine against cutaneous leishmaniasis.

【 授权许可】

CC BY