Construction of expression vectors for secretion of human interferons by yeast.

Publisher Summary This chapter discusses the construction of expression vectors for the secretion of human interferons by yeast. With the advent of heterologous (non-yeast) gene expression in yeast with yeast plasmids and the 5'- and 3'-flanking DNA control regions of highly expressed yeast genes, it is now possible to use heterologous or homologous amino-terminal signal sequences to produce and secrete heterologous proteins from yeast. Using such expression systems, it is demonstrated that signal sequences of two human leukocyte interferons (IFN-αA and IFN-αD) and human gamma interferon (IFN-γ) direct the secretion of these protein products into the periplasm and growth medium of yeast. A variation of previously described protocols is used for oligonucleotide-directed deletion mutagenesis. The single-stranded DNA template is prepared from the recombinant M13mp8 phage containing the appropriate insert. This template is annealed with the phosphorylated synthetic oligonucleotide of 24 bases in length. IFN-α secretion and processing can be obtained with two different homologous yeast signal sequences attached to IFN-α, with one signal producing 80% of yeast medium protein as IFN-αD. The two secretion signals used are obtained from the yeast invertase and α-factor genes.