Microbody phosphoglycerate kinase of Trypanosoma brucei: expression and complementation in Escherichia coli

In the primitive eukaryotic parasite, Trypanosoma brucei, most of the enzymes of glycolysis are located within microbody organelles called glycosomes. Proteins destined for the glycosome are synthesized on free ribosomes and post-translationally translocated into the organelle. The gene, gPGK, encoding the glycosomal isozyme of phosphoglycerate kinase (gPGK), was cloned adjacent to a T7 promoter and cotransformed with a plasmid encoding T7 RNA polymerase into Escherichia coli Pgk-cells. Functional complementation occurred, but only after the creation of a ribosome-binding site by mutagenesis. This represents the first example of complementation of an E. coli mutant with a gene encoding a microbody protein. Enzymatically active recombinant gPGK was purified to near homogeneity by ion exchange chromatography from highly expressing E. coli. The recombinant protein will aid in studies of glycosomal biogenesis.