Expression by Chlamydomonas reinhardtii of a chloroplast ATP synthase with polyhistidine-tagged beta subunits.
The green alga Chlamydomonas reinhardtii is a model organism for the study of photosynthesis. The chloroplast ATP synthase is responsible for the synthesis of ATP during photosynthesis. Using genetic engineering and biolistic transformation, a string of eight histidine residues has been inserted into the amino-terminal end of the beta subunit of this enzyme in C. reinhardtii. The incorporation of these amino acids did not impact the function of the ATP synthase either in vivo or in vitro and the resulting strain of C. reinhardtii showed normal growth. The addition of these amino acids can be seen through altered gel mobility of the beta subunit and the binding of a polyhistidine-specific dye to the subunit. The purified his-tagged CF1 has normal Mg(2+)-ATPase activity, which can be stimulated by alcohol and detergents and the enzyme remains active while bound to a nickel-coated surface. Potential uses for this tagged enzyme as a biochemical tool are discussed.
Johnson
Eric A
EA
Department of Biology, Johns Hopkins University, Baltimore, MD 21218-2685, USA. ejohn@jhu.edu
Rosenberg
Julian
J
McCarty
Richard E
RE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D002736:Chloroplasts / Q000201:enzymology*; D017931:DNA Primers; D006639:Histidine; D007700:Kinetics; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010788:Photosynthesis; D021122:Protein Subunits / Q000737:chemistry; D015800:Protozoan Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D017931:DNA Primers; D021122:Protein Subunits; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D000255:Adenosine Triphosphate; D025261:Mitochondrial Proton-Translocating ATPases