Abstract
ABSTRACTBesides mass drug administration, successful elimination of human lymphatic filariasis, a mosquito-borne tropical parasitic disease, requires developing other suitable prophylactic agents such as vaccines. The Brugia malayi abundant larval transcript-2 (BmALT-2) protein has been identified as one possible candidate. So far, it (E-ALT-2) has been expressed as a 24-kDa non-glycosylated protein in Escherichia coli. Easy and low-cost downstream purification of secreted BmALT-2 in Pichia pastoris may be a vital option to inexpensive large-scale production. This study focused on expression and molecular characterization of BmALT-2 (P-ALT-2) in P. pastoris. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that some P. pastoris colonies produced 27 and 24 kDa bands and few colonies produced a 24-kDa band. Preliminary studies confirmed glycosylation of 27 kDa P-ALT-2. The ratio of glycosylated and non-glycosylated P-ALT-2 was 20:80–70:30 in various colonies. The maximum yield of glycosylated and non-glycosylated P-ALT-2 was measured as 7.99 ± 1.12 and 9.18 ± 1.35 mg L−1 compared to 6.5 ± 1.2 mg L−1 of E-ALT-2 in shake flasks. Their overall expression was about 25% and 35% higher than that in E. coli. The glycosylated P-ALT-2 exhibited about 15% higher immunoreactivity with human endemic normal sera. The enhanced secreted production by P. pastoris may lead to cost-effective large-scale production of BmALT-2.
Highlights
Lymphatic filariasis is a neglected tropical parasitic disease caused mostly by mosquito-borne Wuchereria bancrofti and Brugia malayi
This study focused on expression and molecular characterization of Brugia malayi abundant larval transcript-2 (BmALT-2) (P-abundant larval transcript-2 (ALT-2)) in P. pastoris
Being an L3-specific antigen, the ALT-2 protein is prevalent in endemic normal (EN) sera followed by chronic pathology (CP) and microfilariae (MF)
Summary
Lymphatic filariasis is a neglected tropical parasitic disease caused mostly by mosquito-borne Wuchereria bancrofti and Brugia malayi. Progressive increase in the expression of proteins such as glucoamylase, lipase, green fluorescence protein, influenza hemagglutinin and human serum albumin was achieved with up to seven gene copies [30]. Proteins such as cattle tick vaccine [31], endo-b-1,4-mannase [32] and hepatitis B surface antigen [33,34] have been successfully expressed in P. pastoris. The expression of BmALT-2 was optimized in flask culture This was a new attempt to express this vaccine candidate in a eukaryotic expression system such as P. pastoris. We compared the yield of BmALT-2 in both systems along with its glycosylation followed by their reactivity with human clinical sera samples
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