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Expression of PEPCase cDNA in E. coli with T7 RNA polymerase/promoter system

1993; Elsevier BV; Volume: 19; Issue: 3 Linguagem: Inglês

1671-3877

Dong Longying, Jun Lin, Shi Jiaonai,

Photosynthetic Processes and Mechanisms

Phosphoenolpyruvate carboxylase (PEPCase) is one of the key enzymes in C_(4)-dicarboxylic assimilation, and an important target for genetic manipulation for increasing photosynthetic efficiency in higher plants. For introducing PEPCase gene into C_(3) a plant, we preliminarily selected the T7 RNA polymerase/promoter system, a very usefully expression plasmid, to express PEPCase cDNA in E. coli, and tried to find out the optimum conditions for its expression in higher plants. pPEP3055 plasmid was digested with EcoR I, and 2.0 kb fragment of PEPCase cDNA was recovered by electroelution method, then it was subcloned into a T7 plasmid with T7 RNA polymerase promoter and introduced into E. coli K38 harboring a resident plasmid of pGP1-2. A recombinant clone of PEPCase cDNA (pGTE 1) was screened by Southern (1975) hybridization. As there was a heat-sensitive repressor geneC1857 in appropriate concentration of rifampicin was added to inhibit the expression of RNA polymerase of E. coli, the PEPCase cDNA in recombinant plasmid was effectively expressed. An additional band of protein was observed on the SDS-PAGE, which was heavily contaminated by background proteins. Then Maxcell method was used to identify the expression product. When the sample was pulsed with ~(35)S-Met, two small more clearcut bands appeared on the autoradiographic film, but other newly synthesized proteins also appeared. It shows that there is not high enough expression specificity. In order to increase the speicificity, a concentrations gradient of rifampicin was used, and good results were obtained. Only two additonal protein bands were selectively expressed at a concentration of 200 #mu#g/ml rifampicin. In that case, protein product was more than 90% of the total protein. Western blot test revealed that the two additional bands were subunits of PEPCase with a molecular weight of 78 and 80 kD respectively.