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Bacterial production and purification of recombinant human prolactin
Paris, N.; Rentier-Delrue, Françoise; Defontaine, A. et al.
1990In Biotechnology and Applied Biochemistry, 12 (4), p. 436-49
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Keywords :
Amino Acid Sequence; Base Sequence; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Escherichia coli/*genetics; *Genetic Vectors; Humans; Inclusion Bodies/analysis; Isoelectric Focusing; Molecular Sequence Data; Plasmids/genetics; Prolactin/*biosynthesis/genetics/isolation & purification; Recombinant Proteins/biosynthesis/genetics/isolation & purification; Solubility
Abstract :
[en] Escherichia coli cells transformed with a recombinant plasmid (pT7L) containing the coding sequence of human prolactin (hPrl) expressed a new protein. This protein, comigrating with human Prl on sodium dodecyl sulfate (SDS)-polyacrylamide gels, represented 50% of the total bacterial extract. Immunoprecipitation of [35S]methionine-labeled bacterial lysate with a rabbit antiserum to hPrl followed by SDS-polyacrylamide gel electrophoresis (PAGE) analysis showed that the major component had a Mr identical to that of standard hPrl. The majority of the recombinant hPrl (r-hPrl) accumulated in inclusion bodies. Analysis of these inclusion bodies by SDS-PAGE under nonreducing conditions showed that they are composed mostly of fully reduced monomers. Solubilization of the inclusion bodies and protein denaturation were performed in 8 M urea. Refolding during the renaturation procedure was confirmed by SDS-PAGE under nonreducing conditions. r-hPrl was further purified by gel permeation chromatography on a fast protein liquid chromatography column. More than 95% of the molecules were recovered as oxidized monomeric forms. The refolded molecule was tested for its bioactivity in the Nb2 lymphoma mitogenic assay. The dose-response curves obtained with either r-hPrl or pituitary-derived hPrl showed a complete parallelism. Furthermore, Nb2 cell proliferation was completely blocked by addition of hPrl antiserum to both preparations. Recombinant hPrl is identical to natural hPrl except for an additional methionine group at the amino terminal end.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Paris, N.
Rentier-Delrue, Françoise ;  Université de Liège - ULiège > Département des sciences de la vie > Biologie et génétique moléculaire - GIGA-R : Coordination scientifique
Defontaine, A.
Goffin, V.
Lebrun, J.
Mercier, L.
Martial, Joseph ;  Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire
Language :
English
Title :
Bacterial production and purification of recombinant human prolactin
Publication date :
1990
Journal title :
Biotechnology and Applied Biochemistry
ISSN :
0885-4513
eISSN :
1470-8744
Publisher :
Portland Press, London, United Kingdom
Volume :
12
Issue :
4
Pages :
436-49
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 27 August 2009

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