STRUCTURAL FEATURES OF A POLYPEPTIDE CARRIER PROMOTING SECRETION OF ABETA-LACTAMASE FUSION PROTEIN IN YEAST

Escherichia coli Beta-lactamase was secreted into the culture medium o f Saccharomyces cerevisiae in biologically active form, when fused to the C-terminus of the hsp150 Delta-carrier. The hsp150 Delta-carrier i s an N-terminal fragment of the yeast hsp150 protein, having a signal peptide and consisting mostly of a 19 amino acid peptide repeated 11 t imes in tandem. Here we expressed the hsp150 Delta-carrier fragment al one in S. cerevisiae. Apparently due to a positional effect of the gen e insertion, large amounts of the hsp150 Delta-carrier were synthesize d. About half of the de novo synthesized carrier molecules were secret ed into the culture medium, the rest remaining mostly in the pre-Golgi compartment. The extensively O-glycosylated carrier fragment was puri fied from the culture medium under non-denaturing conditions. Circular dichroism spectroscopy showed that it had no regular secondary struct ure. Nuclear magnetic resonance spectroscopy showed that a non-glycosy lated synthetic peptide, the consensus sequence of the repetitive 19 a mino acid peptide, also lacked secondary structure. The unstructured c arrier polypeptide may facilitate proper folding and secretion of hete rologous proteins attached to it.