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

Abstract

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⁻¹ in the bioreactor. The P-TUFT-ALT-2 expression was measured as about 35 mg L⁻¹, 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.

Keywords:

• Abundant Larval Transcript-2
• bioreactor production
• lymphatic filariasis
• Pichia pastoris; tuftsin

Acknowledgments

We are very thankful to Centre for Biotechnology, Anna University, Chennai for providing research facilities. We acknowledge the heartily support from Dr. K.B. Ramachandran, Indian Institute of Technology, Madras for providing cloning facility.

Disclosure statement

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Additional information

Funding

This project was mainly supported by CSIR-SRF (NET) Fellowship (CSIR sanction no. 09/468(0467)/2012-EMR-1) to Mr. Rajkumar Paul from Council for Scientific and Industrial Research (CSIR), New Delhi. This is also partially funded by Indian Council for Medical Research (ICMR) as ICMR Emeritus Medical Scientist Award to Dr. P. Kaliraj, Centre for Biotechnology, Anna University, Chennai.