ISOLATION AND EXPRESSION OF A GENE SPECIFYING A CDC2-LIKE PROTEIN-KINASE FROM THE HUMAN MALARIA PARASITE PLASMODIUM-FALCIPARUM

A partially redundant oligonucleotide based on conserved protein sequences of cdk and cdc2-like proteins was used to isolate from genomic libraries of Plasmodium falciparum fragments of chromosome XIII carrying a 288-residue open-reading frame encoding a protein kinase sharing 57-58% identity with yeast p34(cdc2). Based on sequence data, base composition and the striking similarity with other cdk and related proteins, four intervening sequences were identified. Their removal in vitro allowed expression of the gene, designated PfPKS, in Escherichia coli, the resulting product having kinase activity against casein and histone H1. Western blotting using a polyclonal antibody raised against the expressed protein showed that the kinase was located in the parasite's cytosol and was present in approximately constant amounts throughout the intra-erythrocytic asexual reproductive stage of the life cycle. The PSTAIRE region of the PfPK5 protein differs at three sites from that of p34(cdc2) and the gene failed to complement cdc2/28 yeast mutants. However, Western blotting showed that the gene was not expressed in yeast, so this does not eliminate the possibility that it is the malarial version of cdc2.