Abstract
Two isozymes of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) of the apicomplexan protozoan Toxoplasma gondii are encoded by the single HGPRT gene as a result of differential splicing. Western blotting of total T. gondii protein shows that both isozymes I and II, which differ by 49 amino acids, are expressed. Both form enzymatically active homotetramers when overexpressed in Escherichia coli. The specific activity of HGPRT-I is five times that of HGPRT-II. When both isozymes are co-expressed in E. coli, HGPRT-I.HGPRT-II heterotetramers form. The predominant heterotetramer has enzymatic activity similar to HGPRT-II, and gel filtration chromatography demonstrates that its size is intermediate between the sizes of HGPRT-I and HGPRT-II. Mass spectrometric analysis of cross-linked homo- and heterotetramers reveals species of distinct molecular mass for HGPRT-I, HGPRT-II, and HGPRT-I.HGPRT-II and suggests that the predominant heterotetramer consists of one HGPRT-I subunit and three HGPRT-II subunits. The implications of this finding are discussed.
Highlights
Encephalitis caused by the apicomplexan protozoan Toxoplasma gondii is the second leading cause of death among patients with AIDS [1]
The cloning of T. gondii hypoxanthine-guanine phosphoribosyltransferase (HGPRT) revealed the presence of two cDNAs, differing by 147 nucleotides, that appear to result from differential splicing of the nascent transcript from the single HGPRT gene [8, 9]
We report here our initial investigations into these questions, which show that T. gondii does express stable HGPRT-II, that the HGPRT-II homotetramer possesses enzymatic activity distinct from that of HGPRT-I, that HGPRT-I and HGPRT-II form a particular heterotetramer when both are co-expressed in Escherichia coli, and that this heterotetramer possesses enzymatic activity similar to that of the HGPRT-II homotetramer
Summary
Subcloning into pET9a—Plasmids pET15b-C1 (previously pETC1; Ref. 8) and pET15b-C3 (pETC3; Ref. 8), which contain the T. gondii HGPRT isozymes I and II coding sequences, respectively, inserted into vector pET15b (Novagen, Inc.; Ref. 14), were digested with NdeI and BamHI overnight at 37 °C. pET9a was digested as well. pET9a is similar to pET15b but lacks the N-terminal His tag and contains a kanamycin rather than an ampicillin resistance marker. PET9a is similar to pET15b but lacks the N-terminal His tag and contains a kanamycin rather than an ampicillin resistance marker. Competent E. coli XL1 Blue cells were transformed to kanamycin resistance with the ligated DNAs and grown on 2x-YT (16 g/liter tryptone, 10 g/liter yeast extract, and 5 g/liter NaCl) agar plates containing 25 g/ml kanamycin. Co-expression of HGPRT-I and HGPRT-II—Competent E. coli BL21(DE3) cells were transformed to simultaneous ampicillin and kanamycin resistance with plasmids pET9a-C1 and pET15b-C3 or plasmids pET9a-C3 and pET15b-C1 (2x-YT agar plates containing 25 g/ml kanamycin and 100 g/ml ampicillin). Single colonies of E. coli BL21(DE3)/ pET9a-C1/pET15b-C3 and BL21(DE3)/pET9a-C3/pET15b-C1 were separately inoculated into 5 ml of 2x-YT broth containing 25 g/ml kanamycin and 100 g/ml ampicillin and grown overnight (250 rpm, 37 °C).
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