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 cDNA library with a synthetic 21-mer oligonucleotide and with rat PRL cDNA. For its expression, use was made of a vector, p3SN8, containing tac-promoter-controlled sequences for a bacterial cellulase leader joined to sequences coding for Ala-Ser, a chromatographic affinity site consisting of six histidines and a Factor Xa cleavage site. The hPRL cDNA was inserted at the 3' end of the cleavage-site sequences. Expression in Escherichia coli led to secretion in the periplasmic space of a fully 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 tag-hPRL could be rapidly and efficiently purified by metal-chelate affinity chromatography. The correct processing and quality of tag-hPRL was monitored by SDS/PAGE, Western-blot analysis, immunoassay and Nb2-lymphoma-cell bioassay. Treatment with Factor Xa for tag removal was only partially successful. Periplasmic secretion of tag-hPRL of the order of 0.7 micrograms/ml per A600 unit and one-step purification indicate feasibility for tag-hPRL production for in vitro diagnostic and research applications. This is the first report describing periplasmic secretion of a bioactive form of hPRL.
Morganti
L
L
National Nuclear Energy Commission (IPEN-CNEN)-Cidade Universitária, São Paulo, Brazil.
Huyer
M
M
Gout
P W
PW
Bartolini
P
P
D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification*; D004926:Escherichia coli / Q000378:metabolism / Q000502:physiology*; D015951:Factor Xa / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011388:Prolactin / Q000096:biosynthesis* / Q000737:chemistry
D018076:DNA, Complementary; C102346:alanyl-seryl-(histidyl)6-isoleucyl-glutamyl-glycyl-arginyl-prolactin; D011388:Prolactin; D015951:Factor Xa