ABSTRACT
The cDNA coding spinach glycolate oxidase (GO) was amplified by RT-PCR using the total RNA of spinach leaves as the template, and was cloned into cloning vector pMD18-T. After the DNA sequence was determined, the go gene was subcloned into E. coli expression vector pBV220, pET-22b(+), pTIG-Trx and pThioHisC. SDS-PAGE analysis revealed that the recombinant E. coli BL21 (DE3) (pTIG-Trx-GO) and E. coli BL21 (DE3) (pET-22b(+)-GO) expressed the predicted 38 kD glycolate oxidase, and the enzyme activity was also detected.
Subject(s)
Alcohol Oxidoreductases , Genetics , Metabolism , Cloning, Molecular , Escherichia coli , Gene Expression , Isopropyl Thiogalactoside , Pharmacology , Spinacia oleracea , GeneticsABSTRACT
In order to establish a method by which the recombinant suicide plasmids integrated on the chromosome could be recircled, A simple method of transconjugative cloning was established with the helper plasmids pMT999 or pRK2013 and fusion strains of Shigella flexneri which were obtained by screening with in vivo expression technology. And the cloning efficiency with this method is very high.