Synthesis, purification and bioactivity of recombinant human activin A expressed in the yeast Pichia pastoris.
The transforming growth factor-beta (TGF-beta) superfamily member, activin A, plays a central role in the regulation of multiple physiological processes including cell differentiation, mitogenesis, embryogenesis, apoptosis and inflammation. In normal cells, activin A signalling is regulated to maintain cellular and tissue health and suppress tumour growth. Disruption of activin A signalling has been implicated in tumour formation and progression. Hence, the availability of activin A is an important target for the development of diagnostics and drugs for therapeutic intervention. To this end, we have expressed human activin A in Pichia pastoris, permitting its secretion into culture medium and purification as the mature homodimer. A construct was engineered encoding the monomeric precursor protein with a N-terminal FLAG affinity tag (DYKDDDDK) and a cleavage site (EKR) for Kex2p protease. Procedures for the two-step purification of human activin A by ion-exchange and anti-FLAG antibody affinity chromatography, and for the removal of the FLAG affinity tag from purified recombinant human activin A by enteropeptidase, are described. The molecular weights of the FLAG-tagged and de-tagged human activin A were confirmed by MALDI-TOF mass spectroscopy. The biological activity of these recombinant activins was assessed for their effects on modulating the secretion of Endothelin-1 (ET-1) by human umbilical vein endothelial cells (HUVECs). The recombinant human activin A containing the intact FLAG tag resulted in a reduced ET-1 secretion from HUVECs, whereas upon removal of this affinity purification tag the purified recombinant human activin A restored ET-1 secretion to levels comparable to the positive control. These results document an approach of considerable potential for the simple, large-scale expression and purification of this important human growth factor for use in diagnostic and therapeutic purposes.
Papakonstantinou
Theo
T
ARC Special Research Centre for Green Chemistry, Building 75, Monash University, Clayton, Victoria 3800, Australia.
Harris
Simon J
SJ
Fredericks
Dale
D
Harrison
Craig
C
Wallace
Euan M
EM
Hearn
Milton T W
MT
D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006801:Humans; D028322:Inhibin-beta Subunits / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011993:Recombinant Fusion Proteins; C103008:inhibin beta A subunit; D028322:Inhibin-beta Subunits