Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner.
Leukemia inhibitory factor (LIF) is a polyfunctional cytokine with numerous regulatory effects in vivo and in vitro. In stem cell cultures it is the essential media supplement for the maintenance of pluripotency of embryonic and induced pluripotent stem cells. With regard to large scale cultures of these cells, LIF is needed in high quality and quantity and represents the major cost determining factor (90%) of the culture media. In this report, we describe a novel production and purification process for human LIF (hLIF) from recombinant Escherichia coli cultures. hLIF was cloned into pET32b and expressed as soluble protein in fusion with thioredoxin. After purification based on membrane adsorber technology, the fusion protein was cleaved using TEV protease. Released, soluble hLIF was subsequently purified by cation exchange chromatography and successfully tested for its biological activity using suspension cultures of murine embryonic and induced pluripotent stem cells. Our novel protocol for the production of recombinant hLIF is very suitable and effective for the production of poorly soluble proteins through expression in fusion with the solubilizing partner thioredoxin.
Tomala
Magda
M
Institute of Technical Chemistry, Leibniz University of Hannover, Callinstr. 5, 30167 Hannover, Germany.
Lavrentieva
Antonina
A
Moretti
Pierre
P
Rinas
Ursula
U
Kasper
Cornelia
C
Stahl
Frank
F
Schambach
Axel
A
Warlich
Eva
E
Martin
Ulrich
U
Cantz
Tobias
T
Scheper
Thomas
T
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D053595:Embryonic Stem Cells; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D053605:Leukemia Inhibitory Factor / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D016256:Lewis X Antigen / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D012995:Solubility; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
D053605:Leukemia Inhibitory Factor; D016256:Lewis X Antigen; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D010450:Endopeptidases; C099488:TEV protease