METHOD FOR SPECIFIC INTEGRATION OF T7 RNA POLYMERASE GENE IN THE CHROMOSOME OF CORYNEBACTERIAL AND THE RESULTANT CORYNEBACTERIA-T7 PROMOTER BASED SHUTTLE VECTOR SYSTEM.

摘要

The present invention relates to method for obtaining optimum expressed proteins in a transformed gram positive bacteria by specific integration of T7 RNA polymerase gene into the chromosome of a gram positive bacteria exhibiting resistance to aminoglycosides, said method comprising:- digesting an E. coli plasmid with a restriction enzyme- digesting the genomic DNA of said gram positive bacteria- ligating the said digested plasmid to the digested genomic DNA of said gram positive bacteria- transforming the said gram positive bacterial protoplasts with the said ligation mixture of step 2 to yield transformed gram positive bacteria (transformants),- screening the said transformants for kanamycin resistance and aminoglycoside sensitivity to ensure that the targetting of the said plasmid vector into the chromosome of the said gram positive bacteria is successful- cloning of the desired gene in the said vector - culturing the transformant in a suitable culture medium- isolating the expressed proteins from the culture medium