[Erythropoietin gene cloning and expression in S. cerevisiae].
Mature human erythropoietin gene was amplified from EPO cDNA by PCR methods. The PCR product was cloned into pUC18 plasmid at Sma I site, then precisely engineered into a intermidiate vector pSK43SB which were digested with Hind III, Mung bean nuclease, and Sal I. Then degest pSK43SB-EPO plasmid with EcoR I and Cla I, the EC fragment with an alpha-factor leading sequence, EPO gene and CYC1 terminater were produced. It was then cloned into a typical high efficiency episomal expression vector YEpHC8. Human EPO protein with highly mannose glycosylated was identified by Western blot methods in both secreted and in cells proteins. N-Glycosidase F digested secreted EPO can produce 20,000 EPO without N-glycosylation similar with that produced in cells.
Zhang
X
X
National Laboratory of Medical Molecular Biology, CAMS, Beijing.
Huang
B
B
Cai
L
L
D000595:Amino Acid Sequence; D000818:Animals; D019677:Artificial Gene Fusion; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004921:Erythropoietin / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D004921:Erythropoietin