The secretion leader of Mucor pusillus rennin which possesses an artificial Lys-Arg sequence directs the secretion of mature human growth hormone by Saccharomyces cerevisiae.
The prepro-peptide of fungal aspartic proteinase, Mucor pusillus rennin, is useful as a secretion leader for efficient secretion of human growth hormone (HGH) from Saccharomyces cerevisiae. For secretion by yeast cells of HGH with the same NH2 terminus as native HGH, an artificial Lys-Arg linker, which is one of the potential KEX2 recognition sequences, was introduced at the junction between the M. pusillus rennin secretion leader and mature HGH. The HGH directed by this construction was the same size as native HGH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid sequencing of its NH2 terminus revealed that the secretion leader peptide was removed correctly at the COOH-terminal side of the Lys-Arg linker. On the other hand, when the same plasmid was expressed in a kex2 mutant strain, unprocessed HGH of a higher molecular weight was secreted, indicating that no proteolytic cleavage at the Lys-Arg site occurred. These results clearly showed that the leader peptide with the Lys-Arg linker was recognized and specifically cleaved by the yeast KEX2 protease. The mature HGH purified from yeast culture medium was indistinguishable from native HGH in biological activity, determined by the adipocyte conversion assay, and in secondary structure, determined by circular dichroism spectroscopy.
Hiramatsu
R
R
Department of Agricultural Chemistry, University of Tokyo, Japan.
Horinouchi
S
S
Uchida
E
E
Hayakawa
T
T
Beppu
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D012085:Chymosin / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004271:DNA, Fungal; D013006:Growth Hormone / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009089:Mucor / Q000235:genetics*; D043484:Proprotein Convertases; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000378:metabolism; D013381:Subtilisins
D004271:DNA, Fungal; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins; D013006:Growth Hormone; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae; D012085:Chymosin