DNA plasmids with improved expression
The present invention relates to the production and use of covalently closed circular (ccc) recombinant plasmids, and more particularly to vector modifications that improve expression of said DNA molecules in the target organism.
1. A method for heat inducible production of a target protein or mRNA gene from the Escherichia coli genome using a temperature sensitive lambda repressor repressed P L promoter, comprising:
a. Cloning the said target protein or mRNA gene into an expression vector to create a P L -target protein or P L -mRNA gene expression cassette in which said target protein or mRNA gene is expressed under the control of the P L promoter and said P L promoter further comprises a OL1 mutation selected from the group comprising P L promoter OL1-G (SEQ ID NO: 11), and P L promoter OL1-G to T (SEQ ID NO: 12);
b. Integrating said P L -target protein or P L -mRNA gene expression cassette into a host strain genome to create a P L -target protein or P L -mRNA host strain in which expression from the said P L -target protein or P L -mRNA gene expression cassette is repressed by a temperature sensitive cl857 lambda repressor;
c. propagating said P L -target protein or P L -mRNA host strain at 25-32° C. to maintain said target protein or mRNA at a basal copy number; and
d. inducing said P L -target protein or P L -mRNA host strain at 37-42° C. to increase expression of said target protein or mRNA.