TY - JOUR

T1 - Structural features of a polypeptide carrier promoting secretion of a beta-lactamase fusion protein in yeast

AU - Jämsä, E

AU - Holkeri, H

AU - Vihinen, H

AU - Wikström, M

AU - Simonen, M

AU - Walse, B

AU - Kalkkinen, N

AU - Paakkola, J

AU - Makarow, M

PY - 1995/11

Y1 - 1995/11

N2 - Escherichia coli beta-lactamase was secreted into the culture medium of Saccharomyces cerevisiae in biologically active form, when fused to the C-terminus of the hsp150 delta-carrier. The hsp150 delta-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 hsp150 delta-carrier fragment alone in S. cerevisiae. Apparently due to a positional effect of the gene insertion, large amounts of the hsp150 delta-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.

AB - Escherichia coli beta-lactamase was secreted into the culture medium of Saccharomyces cerevisiae in biologically active form, when fused to the C-terminus of the hsp150 delta-carrier. The hsp150 delta-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 hsp150 delta-carrier fragment alone in S. cerevisiae. Apparently due to a positional effect of the gene insertion, large amounts of the hsp150 delta-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.

KW - Amino Acid Sequence

KW - Base Sequence

KW - Biological Transport

KW - Carbohydrate Sequence

KW - Escherichia coli

KW - Glycoproteins

KW - Glycosylation

KW - Golgi Apparatus

KW - Heat-Shock Proteins

KW - Molecular Sequence Data

KW - Molecular Weight

KW - Peptide Fragments

KW - Polysaccharides

KW - Protein Structure, Secondary

KW - Repetitive Sequences, Nucleic Acid

KW - Saccharomyces cerevisiae

KW - Saccharomyces cerevisiae Proteins

KW - beta-Lactamases

U2 - 10.1002/yea.320111406

DO - 10.1002/yea.320111406

M3 - Journal article

C2 - 8585321

VL - 11

SP - 1381

EP - 1391

JO - Yeast

JF - Yeast

SN - 0749-503X

IS - 14

ER -