The prepro-peptide of Mucor rennin directs the secretion of human growth hormone by Saccharomyces cerevisiae.
An aspartic proteinase, Mucor pusillus rennin (MPR), of filamentous fungus Mucor pusillus, is efficiently secreted from a transformant of Saccharomyces cerevisiae containing the intact MPR gene. To test the usefulness of the MPR leader peptide in secretion of heterologous proteins from yeast cells, several plasmids encoding the fusion proteins composed of different parts of the NH2-terminal region of prepro-MPR and human growth hormone (hGH) were constructed. The parts of the leader peptide upstream of hGH were the whole prepro-peptide following the NH2-terminal region of mature MPR in JGH1, the intact pre-sequence and a part of the pro-sequence in JGH2, and the putative signal sequences of the NH2-terminal 18 and 22 amino acids in JGH3 and JGH7, respectively. When the hGH genes fused to these leader sequences were expressed in yeast cells under the control of the yeast GAL7 promoter, proteins of various sizes immunoreactive with the anti-hGH antibody were secreted into the medium. Among the plasmids mentioned above, JGH2 directed the greatest secretion of the protein of 23 kilodaltons in size, which contained the expected NH2-terminal amino acid sequence of an additional eight amino acids derived from the pro-peptide of MPR. The addition of the GAL10 terminator downstream of the hGH gene in JGH2 resulted in a greater than three- to fivefold increase in the secretion, whereas the insertion of the GAL4 gene, which is a positive regulator for the GAL system, had no significant effect. The improved yield of the total protein of hGH secreted into the medium reached approximately 10 mg/liter.
Hiramatsu
R
R
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.
Yamashita
T
T
Aikawa
J
J
Horinouchi
S
S
Beppu
T
T
D000595:Amino Acid Sequence; D012085:Chymosin / Q000235:genetics / Q000378:metabolism*; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D005822:Genetic Vectors; D013006:Growth Hormone / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009089:Mucor / Q000201:enzymology / Q000235:genetics; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D004792:Enzyme Precursors; D021382:Protein Sorting Signals; D013006:Growth Hormone; C034675:preprorennin; D012085:Chymosin