Cloning, large-scale production and characterization of fusion protein (P-TUFT-ALT-2) of Brugian abundant larval transcript-2 with tuftsin in Pichia pastoris.

Lymphatic filariasis is a "disease of poor people" due to a large section of affected people with economic backwardness. Therefore, successful elimination of this disease requires a cost-effective prophylactic agent such as vaccine along with conventional drugs. The Abundant Larval Transcript-2 (BmALT-2) protein of Brugia malayi has been recognized as the most potential vaccine candidate. Tuftsin, a tetra-peptide immunopotentiator has already shown the enhanced immunogenicity of various vaccine antigens in earlier studies. This study deals with the development of tuft-alt-2 fusion construct and a suitable culture condition for its large-scale production in Pichia pastoris. The recombinant P. pastoris/tuft-alt-2 with 9-11 copies of the gene construct exhibited the highest expression level. The molecular weight of P-TUFT-ALT-2 was determined as 28 kDa in SDS-PAGE including 3 kDa due to glycosylation. The dry cell biomass was 57.4 gL in the bioreactor. The P-TUFT-ALT-2 expression was measured as about 35 mg L, which was 102% higher than flask culture. The P-TUFT-ALT-2 produced the highest 65,000 IgG peak titer in Balb/c mice. Moreover, P-TUFT-ALT-2 exhibited about 9.46% higher splenocyte proliferation than E. coli expressed E-ALT-2 alone. The enhanced secreted production of P-TUFT-ALT-2 in bioreactor would step up its commercialization as an inexpensive commercial vaccine for human lymphatic filariasis.