SUMMARY: A hybrid plasmid. pDB2, was constructed by ligating a 3.24 kb RI/dIII fragment of the chromosome into pBR322. This was used to transform a. mutant which was devoid of citrate synthase activity. The resultant strain expressed very high citrate synthase activity and this enabled a simplified purification of the homogeneous enzyme in high yield. The subunit was estimated as 47000-49000 by SDS gel electrophoresis, which closely resembles the eukaryotic form of the enzyme. Evidence for some conservation of sequence between the two proteins was revealed in the acid cleavage pattern at aspartyl-prolyl residues. In addition to coding for the structural gene for citrate synthase, the 3.24 kb RI/dIII fragment also retained the genetic structure necessary for control of enzyme synthesis since the expression of enzyme activity in the strain harbouring pDB2 was still subject to glucose repression.


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