ISSN: 0973-7510

E-ISSN: 3373-3386

Anil K. Singh1, Saurabh Shrivastava1, M. Kameswara Rao2, Kshitij chandel3, Hotam singh Chaudhary4, 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.
J. Pure Appl. Microbiol., 2015, 9 (4): 3373-3386.
© The Author(s). 2015
Received: 04/06/2015 | Accepted: 10/08/2015 | Published: 31/12/2015
Abstract

The C-terminal portion of Plasmodium falciparum Merozoite Surface Protein142kDa known to have prophylactic potential was expressed using Pichia pastoris. 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 multicopy 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 via immunoblotting 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 Pichia transformant 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, Pichia pastoris, Secreted expression, multi-copy integrants In – vitro parasite growth inhibition.

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© The Author(s) 2015. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.