PRODUCTION AND CHARACTERIZATION OF BIOLOGICALLY-ACTIVE ALA-SER-(HIS)(6)-ILE-GLU-GLY-ARG-HUMAN PROLACTIN (TAG-HPRL) SECRETED IN THE PERIPLASMIC SPACE OF ESCHERICHIA-COLI

Human prolactin (hPRL) cDNA was obtained by screening of a pituitary c DNA library with a synthetic 21-mer oligonucleotide and with rat PRL c DNA. For its expression, use was made of a vector, p3SN8, containing t ac-promoter-controlled sequences for a bacterial cellulase leader join ed to sequences coding for Ala-Ser, a chromatographic affinity site co nsisting of six histidines and a Factor Xa cleavage site. The hPRL cDN A was inserted at the 3' end of the cleavage-site sequences. Expressio n in Escherichia coil led to secretion in the periplasmic space of a f ully bioactive hPRL variant constituting authentic hPRL with a peptide tag, i.e. Ala-Ser-(His)(6)-Ile-Glu-Gly-Arg, at its N-terminal. This t ag-hPRL could be rapidly and efficiently purified by metal-chelate aff inity chromatography. The correct processing and quality of tag-hPRL w as monitored by SDS/PAGE, Western-blot analysis, immunoassay and Nb2-l ymphoma-cell bioassay. Treatment with Factor Xa for tag removal was on ly partially successful. Periplasmic secretion of tag-hPRL of the orde r of 0.7 mu g/ml per A(600) unit and one-step purification indicate fe asibility for tag-hPRL production for in vitro diagnostic and research applications. This is the first report describing periplasmic secreti on of a bioactive form of hPRL.