Journal of Pure and Applied MicrobiologyVol. 9 No. 4

Heterologous Expression and Characterization of Plasmodium falciparum Merozoite Surface Protein 1 (MSP142kDa) in Pichiapastorisand its in-vitro Parasite Growth Inhibition Assay

Anil K. Singh1, SaurabhShrivastava1, M. KameswaraRao2, Kshitijchandel3, HotamsinghChaudhary4, Prativa K Behara5, C.R. Pillai6 and N Gopalan7*

1Bio-process Scale up Facility, Defence Research and Development Establishment, Ministry of Defence (Govt. of India), Gwalior - 474002, India. 2Biochemistry Division, Defence Research and Development Establishment, Ministry of Defence (Govt. of India), Gwalior - 474002, India. 3ISPAT General Hospital, Rourkela - 769005, Odisha, India. 4Madhav Institute of Technology and Science, Gwalior, India 5National Institute of Malaria Research, Delhi - 110009, India. 6Vector Management Division, Defence Research and Development Establishment, Ministry of Defence (Govt. of India), Gwalior - 474 002, India. 7F.B.D, DFRL, Siddarthanagar, Mysore- 570 011 Ministry of Defence (Govt. of India), Gwalior - 474 002, India.

Received on 04 June 2015 and accepted on 10 August 2015

 

ABSTRACT

The C-terminal portion of Plasmodium falciparumMerozoite Surface Protein142kDa known to have prophylactic potential was expressed using Pichiapastoris. Bioinformatics based B-cell and T-cell epitope predictions revealed uniform distribution of epitopes on MSP142kDa protein. In order to have a broad spectrum protection with multiple epitopes, the complete MSP142kDa was selected for expression using P. pastoris. The gene fragment encoding the MSP142kDa was amplified from the P. falciparum genomic DNA and cloned into pPIC9K yeast transfer vector under the control of AOX1 promoter in fusion with the alpha secretory signal. The expression cassette was integrated into the Pichia genome via homologous recombination. Recombinant Pichia clones carrying multi-copy integrants of the transgene were selected based on geneticin tolerance. The positive Pichia clone was methanol induced to express the transgene and the expression of the recombinant MSP142kDa was confirmed viaimmunoblotting and MALDI-TOF analysis. Purification strategy were developed and verified by SDS-PAGE electrophoresis. Immunization of rabbit with recombinant MSP142kDa in Freund’s adjuvant resulted in high antibody titers against the recombinant MSP-142kDa. Rabbit produced anti-MSP1IgG were inhibited in vitro parasite growth, demonstrating that this inhibition anti-MSP1IgG mediated. The multi-copy recombinant Pichiatransformant obtained in the present study has an immense industrial application for large scale production of MSP142kDa recombinant protein for either prophylactic or diagnostic applications.

Keywords : Plasmodium falciparum, MSP142kDa, Pichiapastoris, Secreted expression, multi-copyintegrantsIn - vitro parasite growth inhibition