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Abstract

The invention describes a stable immunogenic protein having multiple cysteines molecules wherein the protein is having stability up to two years and purity more than 98% particularly rPvRII and/or rPfF2. It also discloses a method for producing said immunogenic protein comprising the following steps: culturing the host E. coli cells containing a desired recombinant gene construct comprising a codon optimized gene sequence of rPvRII and/or rPfF2 to produce cells in high density; inducing expression rPvRII and/or rPfF2 as inclusion bodies; harvesting the cells and isolating the said inclusion bodies; separating rPvRII and/or rPfF2 from inclusion bodies by repeated sequential washing and solubilizing with chaotrophic agents comprising guanidine hydrochloride and/or urea; purifying the protein by subjecting to metal-chelate affinity chromatography; re-folding of the purified rPvRII and/or rPfF2 obtained in step e) with a redox system to recover a high yield of the soluble protein, followed by further purifying the desired protein by removing impurities by subjecting to chromatography. Further the invention discloses formulation comprising rPvRII or rPfF2, preferably being lyophilized using polysaccharides preferably sucrose, lactose, and pharmaceutically acceptable adjuvants such as aluminum hydroxide, aluminum phosphate, CpG nucleotides, non-CpG nucleotides, Montanide ISA-720, MF-59, Mono-phosphoryl Lipid-A (MPL-A) and QS-21.

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First Claim