Synthesis of glia-derived nexin in yeast.
Glia-derived nexin (GDN) is a 43-kDa glycoprotein isolated from rat glioma cell cultures. It promotes neurite extension in cultures of neuroblastoma cells and chick sympathetic neurons. Moreover, GDN is a potent serine protease inhibitor (serpin), belonging to the family of protease nexins. We report here the expression of rat GDN in the Saccharomyces cerevisiae strain GRF18 under the control of the PHO5 promoter. We describe the purification of more than 6 mg total GDN from the cellular extract of 1 liter of yeast culture. The amino acid composition and the sequence of CNBr-fragments of the recombinant protein correlate with the values deduced from the rat GDN cDNA. We provide evidence that the recombinant GDN has exactly the same properties as the glioma-derived protein with respect to its protease-inhibitory activity and its ability to promote the extension of neurites from neuroblastoma cells. The large amounts of recombinant protein obtained from this expression system will allow further biochemical and physiological analysis of GDN and of the serpins in general.
Sommer
J
J
Friedrich Miescher Institute, Basel, Switzerland.
Meyhack
B
B
Rovelli
G
G
Buergi
R
R
Monard
D
D
D016564:Amyloid beta-Protein Precursor; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005910:Glioma; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D011480:Protease Inhibitors / Q000378:metabolism*; D058255:Protease Nexins; D051381:Rats; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D013104:Spheroplasts / Q000378:metabolism
D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D015345:Oligonucleotide Probes; D011480:Protease Inhibitors; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins