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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
Author(s) -
Morganti L.,
Huyer M.,
PW Gout,
Bartolini P.
Publication year - 1996
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1996.tb00365.x
Subject(s) - periplasmic space , complementary dna , escherichia coli , microbiology and biotechnology , biology , cdna library , affinity chromatography , biochemistry , secretion , expression vector , recombinant dna , cleavage (geology) , enzyme , gene , paleontology , fracture (geology)
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.