Complementation of fragments of triosephosphate isomerase defined by exon boundaries.
Chicken triosephosphate isomerase (TIM) has been fragmented by inserting single "splits" at three separate exon/exon boundaries, and the complexes have been assayed for catalytic activity. In vivo studies show that the expression of both portions of each of the three different split genes complements the TIM deficiency of Escherichia coli strain DF502 when grown on selective media. The expression of only one fragment of each split gene does not complement the TIM-minus genotype. To assess the catalytic activity that derives from the fragmented protein complex, the individual peptide products of one of the three split genes were expressed and purified. The purified complex showed isomerase activity, albeit of low specific catalytic activity. A catalytically active multichain complex composed of separate peptide products of a gene singly split at exon/exon junctions has thus been created.
Bertolaet
B L
BL
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138, USA.
Knowles
J R
JR
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002645:Chickens; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005091:Exons; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014305:Triose-Phosphate Isomerase