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Structural features of a polypeptide carrier promoting secretion of a β‐lactamase fusion protein in yeast
Author(s) -
Jämsä Eija,
Holkeri Heidi,
Vihinen Helena,
Wikströum Mats,
Simonen Marjo,
Walse Björn,
Kalkkinen Nisse,
Paakkola Juha,
Makarow Marja
Publication year - 1995
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.320111406
Subject(s) - biology , saccharomyces cerevisiae , circular dichroism , biochemistry , signal peptide , peptide , amino acid , fusion protein , escherichia coli , secretion , peptide sequence , yeast , heterologous , protein secondary structure , microbiology and biotechnology , recombinant dna , gene
Abstract Escherichia coli β‐lactamase was secreted into the culture medium of Saccharomyces cerevisiae in biologically active form, when fused to the C‐terminus of the hsp150δ‐carrier. The hsp150δ‐carrier is an N‐terminal fragment of the yeast hsp150 protein, having a signal peptide and consisting mostly of a 19 amino acid peptide repeated 11 times in tandem . Here we expressed the hsp150δ‐carrier fragment alone in S. cerevisiae . Apparently due to a positional effect of the gene insertion, large amounts of the hsp150δ‐carrier were synthesized. About half of the de novo synthesized carrier molecules were secreted into the culture medium, the rest remaining mostly in the pre‐Golgi compartment. The extensively O‐glycosylated carrier fragment was purified from the culture medium under non‐denaturing conditions. Circular dichroism spectroscopy showed that it had no regular secondary structure. Nuclear magnetic resonance spectroscopy showed that a non‐glycosylated synthetic peptide, the consensus sequence of the repetitive 19 amino acid peptide, also lacked secondary structure. The unstructured carrier polypeptide may facilitate proper folding and secretion of heterologous proteins attached to it.