Method for expression of heterologous proteins in yeast

A method for expression of a heterologous protein and its secretion in the biologically active mature form using a yeast host cell from the genus Kluyveromyces as the expression system, said method comprising transforming said yeast cell with a vector comprising a nucleotide sequence that comprises in the 5′ to 3′ direction and operably linked (a) a yeast-recognized transcription and translation initiation region, (b) a coding sequence for a hybrid precursor polypeptide, and (c) a yeast-recognized transcription and translation termination region, wherein said hybrid precursor polypeptide comprises:

• 5′-SP-(PS)n−1-(LP-PS)n−2-(NPROMHP-PS)n−3-MHP-(PS-CPROMHP)n−4-3′
• wherein:
• SP comprises a signal peptide sequence for a yeast secreted protein;
• PS comprises a preferred processing site cleaved in vivo by a yeast proteolytic enzyme;
• LP comprises a leader peptide sequence for a yeast secreted protein;
• NPROMHP comprises a native N-terminal propeptide sequence of a mature heterologous mammalian protein of interest;
• MHP comprises a peptide sequence for said mature heterologous mammalian protein of interest;
• CPROMHP comprises a native C-terminal propeptide sequence of said mature heterologous mammalian protein of interest;
• and
• n−1, n−2, n−3, and n−4 independently=0 or 1;
• wherein said mammalian protein is a native PDGF-BB protein or variant thereof, wherein said variant has an amino acid sequence that has at least about 80% sequence identity to the amino acid sequence of said native PDGF-BB protein, wherein said processing sites allow for proteolytic processing of said precursor polypeptide to said mature protein in vivo by said yeast host cell, and wherein at least n−3 or n−4=1;
• and culturing said transformed cell such that said protein is expressed.