Secretion by Saccharomyces cerevisiae of rat apolipoprotein E as a fusion to Mucor rennin.
As the first step for production of rat apolipoprotein E (rApoE) in Saccharomyces cerevisiae, the rApoE cDNA was cloned and its nucleotide sequence was determined. When the intact rApoE gene including the presequence-encoding region was expressed under the control of the yeast GAL7 promoter, no protein immunoreactive with anti-rApoE antibody was detected either in the culture medium or inside the cells. For the purpose of the extracellular production of rApoE, three fusion genes were constructed in which the mature rApoE-encoding sequence was connected after the pre, prepro, and whole regions of the gene encoding a fungal aspartic proteinase, Mucor pusillus rennin (MPP), since MPP is efficiently secreted from recombinant S. cerevisiae containing the MPP gene. When these three fusion genes were expressed under the control of the GAL7 promoter, only one, encoding the mature rApoE connected to the whole MPP sequence, directed efficient secretion of the fused protein. The maximum yield of the fused protein secreted into the medium reached 11.8 mg/l and the calculated rApoE part was 5.3 mg in the fused protein. The excreted fusion protein was glycosylated at the original two sites in the MPP part. The fused protein was gradually degraded in the medium probably by proteases of the host cell, because no such degradation occurred in a yeast pep4mutant strain.
Nomura
N
N
Department of Biotechnology, University of Tokyo, Japan.
Yamada
H
H
Matsubara
N
N
Horinouchi
S
S
Beppu
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001057:Apolipoproteins E / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D016282:Aspartic Acid Endopeptidases / Q000235:genetics; D001483:Base Sequence; D012085:Chymosin / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005800:Genes, Fungal; D005822:Genetic Vectors; D006031:Glycosylation; D008969:Molecular Sequence Data; D009089:Mucor / Q000235:genetics*; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*
D001057:Apolipoproteins E; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases; D012085:Chymosin