Biotechnology and Applied Biochemistry (1990) 12, (436–449) (Printed in Great Britain)

Send to a friend Bacterial production and purification of recombinant human prolactin.

Paris N, Rentier-Delrue F, Defontaine A, Goffin V, Lebrun JJ, Mercier L, Martial JA

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INSERM U 298, Universite d'Angers, CHRU, France.

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.

The Biochemical Society, London © 1990

Biotechnology and Applied Biochemistry (1996) 23, (67–75) (Printed in Great Britain)
Send to a friend 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.
Morganti L, Huyer M, Gout PW, Bartolini P

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National Nuclear Energy Commission (IPEN-CNEN)-Cidade Universitaria, Sao Paulo, Brazil.

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.

The Biochemical Society, London © 1996

Biotechnology and Applied Biochemistry (2006) 45, (1–12) (Printed in Great Britain)
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Add a comment Review article
Bioreactor systems for the production of biopharmaceuticals from animal cells
James N. Warnock* and Mohamed Al-Rubeai†1
*Department of Agricultural and Biological Engineering, Mississippi State University, Mississippi
State, MS 39762, U.S.A., and †School of Chemical and Bioprocess Engineering, University
College Dublin and Center for Synthesis and Chemical Biology, Belfield, Dublin 4, Ireland

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Key words: animal cell biotechnology, biopharmaceuticals, bioreactor system, filtration system,
microcarrier, perfusion culture.

Abbreviations used: ATF, alternating tangential flow; BHK cell, baby-hamster kidney cell; CHO
cell, Chinese-hamster ovary cell; ECS, extracapillary space; EPO, erythropoietin; HFBR, hollow-
fibre bioreactor; ICS, intracapillary space; IFN-g, interferon-g; IL-2, interleukin-2; IP, infectious
particles; mAb, monoclonal antibody; OUR, oxygen uptake rate; STR, stirred tank reactor.

1To whom correspondence should be addressed (email m.al-rubeai@ucd.ie).

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The demand for biopharmaceutical products is set to see a significant increase over the next few
years. As a consequence, the processes used to produce these products must be able to meet market
requirements. The present paper reviews the current technologies available for animal cell culture
and highlights the advantages and disadvantages of each method, while also providing details of
recent case studies. Processes are described for both suspension and anchorage-dependent cell lines.

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Received 6 December 2005/21 March 2006; accepted 28 March 2006

Published on the Internet 20 June 2006, doi:10.1042/BA20050233