High-level secretion of human alpha 1-antitrypsin from Saccharomyces cerevisiae using inulinase signal sequence.
The use of a proper signal sequence is one of the major determinants for the efficient secretion of heterologous proteins from yeast. The signal sequence derived from inulinase (INU1A) of Kluyveromyces marxianus was evaluated in directing the secretion of a human glycoprotein, alpha 1-antitrypsin (alpha 1-AT), from Saccharomyces cerevisiae. A yeast expression vector for alpha 1-AT was constructed by placing the coding sequence of human alpha 1-AT fused with the INU1A signal sequence downstream of the GAL10 promoter. S. cerevisiae transformants harboring the expression vector secreted about 70% of the total alpha 1-AT synthesized into the culture media. The intracellularly retained form of alpha 1-AT was mostly unglycosylated, whereas the secreted protein had high mannose-type glycosylation. The fed-batch cultivation of the recombinant yeast achieved a high-cell density, leading to the secretion of biologically active alpha 1-AT up to 75 mg l-1. The secreted protein was purified and subjected to N-terminal sequencing, which confirmed that the secreted alpha 1-AT was processed correctly at the Kex2 cleavage site as expected from the sequence of INU1A signal peptide. The results suggest that the inulinase signal sequence is useful for the high-level secretion of relatively large glycoproteins, such as human alpha 1-AT, from S. cerevisiae.
Kang
H A
HA
Korea Research Institute of Bioscience and Biotechnology, KIST, Taejon, Korea.
Nam
S W
SW
Kwon
K S
KS
Chung
B H
BH
Yu
M H
MH
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001709:Biotechnology; D004274:DNA, Recombinant / Q000235:genetics; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D007716:Kluyveromyces / Q000235:genetics; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D004274:DNA, Recombinant; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000515:alpha 1-Antitrypsin; D006026:Glycoside Hydrolases; C023086:inulinase