EFFICIENT SECRETION IN YEAST BASED ON FRAGMENTS FROM K1 KILLER PREPROTOXIN

被引:14
作者
CARTWRIGHT, CP [1 ]
ZHU, YS [1 ]
TIPPER, DJ [1 ]
机构
[1] UNIV MASSACHUSETTS,SCH MED,DEPT MOLEC GENET & MICROBIOL,WORCESTER,MA 01655
关键词
SACCHAROMYCES-CEREVISIAE; KILLER YEAST; PROTEIN SECRETION; HETEROLOGOUS GENE EXPRESSION;
D O I
10.1002/yea.320080404
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The alpha and beta-components of the secreted K1 killer toxin of Saccharomyces cerevisiae are derived from residues 45-147 and 234-316, respectively, of the 316 residue preprotoxin (ppTox). The beta-N-terminus is produced by Kex2 cleavage after Lys Arg233; when beta-la (the mature sequence of beta-lactamase) is fused at this site and the fusion is expressed from the PGK promoter in pDT 17, a multicopy plasmid, unexpectedly modest levels of beta-la secretion resulted. Over-expression of Kex2 failed to increase beta-la secretion while a kex2-null mutation reduced secretion by 98%. Beta-la secretion in a Kex+ strain was not enhanced by inactivation of the alpha-toxin component or by deletion of most of its central hydrophobic segments. However SP-beta-la, produced by deletion of ppTox residues 35-176, expressed 10-fold higher beta-la activity and the precursor was now secreted with similar efficiency in a kex2-null strain. Fusions of beta-la to ppTox at Ala34 or Ala46 also led to efficient secretion in both KEX2 and kex2-null strains. Since these beta-la fusions differ only in segments well downstream of the signal peptide and all had similar transcript levels, the efficiency of beta-la secretion is apparently determined by the efficiency with which these fusions are translocated to the Golgi compartment where Kex2 is active. Efficiency is high for the shorter fusions, but is 10% or less for the longer fusions; even this fraction is apparently diverted to the vacuole if not cleaved by Kex2. SP-beta-la was the most efficient construct tested; secreted beta-la reached 4% of total cell protein, modestly exceeding levels produced by fusion to the MF-alpha-1-encoded prepro-alpha-factor, suggesting potential for the production of foreign proteins in yeast.
引用
收藏
页码:261 / 272
页数:12
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