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

• Published in: Preparative biochemistry & biotechnology Vol. 48; no. 9; pp. 823 - 833
• Main Authors: Paul, Rajkumar, Karthik, Selvarajan, Vimalraj, Ponnusamy, Meenakshisundaram, Sankaranarayanan, Kaliraj, Perumal
• Format: Journal Article
• Language: English
• Published: England Taylor & Francis 21-10-2018; Taylor & Francis Ltd
• Subjects: Abundant Larval Transcript-2; Animals; Antigens; Antigens, Helminth - biosynthesis; Antigens, Helminth - chemistry; Antigens, Helminth - genetics; Antigens, Helminth - immunology; Base Sequence; bioreactor production; Bioreactors; Brugia malayi - chemistry; Cell culture; Cloning; Cloning, Molecular - methods; Commercialization; Dry cells; E coli; Filariasis; Fusion protein; Gel electrophoresis; Gene expression; Glycosylation; Immunogenicity; Immunoglobulin G; Immunologic Factors - biosynthesis; Immunologic Factors - chemistry; Immunologic Factors - genetics; Immunologic Factors - immunology; lymphatic filariasis; Male; Mice, Inbred BALB C; Molecular weight; Pichia - genetics; Pichia - metabolism; Pichia pastoris; Pichia pastoris; tuftsin; Protein Processing, Post-Translational; Proteins; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Sodium lauryl sulfate; Transcription; Tuftsin - biosynthesis; Tuftsin - chemistry; Tuftsin - genetics; Tuftsin - immunology; Vaccines; Vector-borne diseases; Yeast; lymphatic filariasis; bioreactor production; Pichia pastoris tuftsin; Abundant Larval Transcript-2
• ISSN: 1082-6068; 1532-2297
• DOI: 10.1080/10826068.2018.1514511

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.