Effect of the truncation of the C-terminal region of Kex2 endoprotease on processing of the recombinant Rhizopus oryzae lipase precursor in the co-expression system in yeast

The Saccharomyces cerevisiae Kex2p is a membrane-bound endoprotease and cleaves at a C-terminal site of Lys–Arg of the α-factor precursor in late Golgi compartment. The complete cleavage of a secreted recombinant Rhizopus oryzae lipase (ROL) precursor (rProROL) having an internal Lys–Arg has been examined by the co-expression of the KEX2 gene. The cleavage of rProROL was not sufficient. The co-expression system of the truncated gene encoding the soluble form of Kex2p (sKex2p) lacking C-terminal 201 amino acids containing the transmembrane domain (TMD) was constructed. sKex2p co-expressed was detected in the culture medium and the carboxyl site of Lys(-30)–Arg(-29) in the prosequence of rProROL was completely cleaved. These results revealed that on the process of secretion, including in the Golgi apparatus and secretion vesicles, attack of sKex2p to the produced protein occurred more efficiently than that of Kex2p. The cells having the co-expression system of the gene encoding sKex2p will be useful for production of foreign proteins designed to cleave the internal Lys–Arg site for their activation or for the application of the prepro-α-factor leader sequence.