Improved methods for maximizing expression of a cloned gene: a bacterium that synthesizes rabbit beta-globin.
In this paper we describe a method for constructing E. coli plasmids that direct efficient expression of genes that encode eucaryotic or procaryotic proteins. No functional assays for the proteins are needed, and they are produced in their native, unfused state. The only requirement is that the genes be isolable without intervening sequences. We describe as an example the construction of a plasmid that directs the synthesis of about 10,000-15,000 monomers per cell of rabbit beta-globin. The essential steps in a typical construction are as follows. --A region of the gene encoding the amino-terminal portion of the protein is fused to DNA encoding an enzymatically active carboxy terminal fragment of beta-galactosidase. The latter is carried on one of three plasmids designed to facilitate the fusion (the construction of these three plasmids is described in the Appendix). --A "portable promoter" of the lac operon is placed at many positions in front of the fused gene using nucleases in vitro. Those promoter placements that elicit efficient expression of the fused gene are identified by the beta-galactosidase activity that they express. (In the special case we describe, plasmids identified as directing efficient expression of beta-globin were found to bear "hybrid" ribosome binding sites consisting of the Shine-Dalgarno sequence carried on the promoter fragment and the ATG of the beta-globin gene.) --The gene of interest is reconstituted intact, with the portable promoter in place, by recombination in vitro or in vivo.
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005914:Globins / Q000096:biosynthesis / Q000235:genetics*; D007763:Lac Operon; D010957:Plasmids; D011817:Rabbits; D001616:beta-Galactosidase / Q000096:biosynthesis
D004274:DNA, Recombinant; D005914:Globins; D004262:DNA Restriction Enzymes; D001616:beta-Galactosidase