Structural features of a polypeptide carrier promoting secretion of a beta-lactamase fusion protein in yeast.
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.
Jämsä
E
E
Institute of Biotechnology, University of Helsinki, Finland.
Holkeri
H
H
Vihinen
H
H
Wikström
M
M
Simonen
M
M
Walse
B
B
Kalkkinen
N
N
Paakkola
J
J
Makarow
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002240:Carbohydrate Sequence; D004926:Escherichia coli / Q000201:enzymology; D006023:Glycoproteins; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011134:Polysaccharides / Q000032:analysis; D017433:Protein Structure, Secondary; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D001618:beta-Lactamases / Q000378:metabolism
D006023:Glycoproteins; C074229:HSP150 protein, S cerevisiae; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D011134:Polysaccharides; D029701:Saccharomyces cerevisiae Proteins; D001618:beta-Lactamases