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 f or the efficient secretion of heterologous proteins from yeast. The si gnal sequence derived from inulinase (INU1A) of Kluyveromyces marxianu s was evaluated in directing the secretion of a human glycoprotein, al pha(1)-antitrypsin (alpha(1)-AT), from Saccharomyces cerevisiae. A yea st expression vector for alpha(1)-AT was constructed by placing the co ding sequence of human alpha(1)-AT fused with the INU1A signal sequenc e downstream of the GAL10 promoter. S. cerevisiae transformants harbor ing 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 secret ion of biologically active alpha(1)-AT up to 75 mgl(-1). The secreted protein was purified and subjected to N-terminal sequencing, which con firmed that the secreted alpha(1)-AT was processed correctly at the Ke x2 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.