binding site competent cells of strain c600 were transformed with each ligation reaction plasmid dna was isolated from several drug resistant colonies from each transformation and was subjected to restriction endonuclease digestion analyses the trp promoter containing fragment could insert into the pre expression plasmid in two possible orientations relative to the direction of transcription from the trp promoter the orientation of the promoter insert fragment which would result in the transcription of the pgh gene generates a 920 bp hindiii dna fragment while the undesired orientation generates a 600 bp dna fragment multiple isolates from each ligation reaction were identified with plasmids bearing the complete pgh gene adjacent to the trp promoter and rbs sequence in the configuration required for direct expression further characterization of these expression constructions was achieved by physical mapping with additional restriction endonucleases additional characterization of these pgh expression plasmids by dna sequencing has established their separate identity at the nucleotide level the two pgh expression plasmids were designated pgh 101 and pgh 107 translation of the dna sequence of these pgh expression plasmids predicts a hormone polypeptide of 191 amino acids the estimated molecular weight of such a protein would be about 22 000 when recombinant microorganisms comprising plasmids pgh 101 or pgh 107 were grown under conditions known to induce trp promoter directed expression the cells produced a protein with a size of about 22 000 daltons this band was absent from protein extracts prepared from cells containing the vector plasmid pbr322 grown under identical conditions the level of pgh production was determined using densitometer scans of individual lanes of sds polyacrylamide gels several e coli proteins fall.