High level secretion by Saccharomyces cerevisiae of human apolipoprotein E as a fusion to Rhizomucor rennin.
As the first step for production of human apolipoprotein E (hApoE) in Saccharomyces cerevisiae, the hApoE cDNA was cloned in Escherichia coli, on the basis of the nucleotide sequence reported previously. When the hApoE cDNA including its pre-sequence-encoding region was expressed under the control of the GAL7 promoter, no protein immunoreactive with anti-hApoE antibody was detected either in the culture medium or inside the cells. For efficient production and secretion of hApoE in S. cerevisiae, the mature hApoE-encoding region was fused to the prepro-sequence region of Rhizomucor rennin (MPR) and to the whole MPR gene including its prepro- and mature-MPR regions. When the fusion gene consisting of the prepro-sequence-encoding region and hApoE regions was expressed in S. cerevisiae, no protein reactive with the anti-hApoE antibody was detected in any fraction of the yeast cells, probably due to rapid degradation of the hApoE protein by yeast proteases. On the other hand, when hApoE was expressed as a fusion to the whole MPR protein, a considerable amount of the fused protein was secreted into the medium. The prepro-sequence of MPR was correctly processed from the fused protein in the medium by autocatalytic activity of MPR and by a protease(s) of the host cell.(ABSTRACT TRUNCATED AT 250 WORDS)
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; D001057:Apolipoproteins E / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D012085:Chymosin / Q000378:metabolism*; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009090:Mucorales / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D001057:Apolipoproteins E; D011994:Recombinant Proteins; D012085:Chymosin