Emi Mitsuru

Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens

Abstract Two cDNA clones encoding two major human trypsinogen isozymes were isolated from a human pancreatic cDNA library. The deduced amino acid (aa) sequences of the two trypsinogen precursors are found to have 89% sequence homology, and have the same number of aa (247), including 15 aa for a signal peptide and 8 aa for an activation peptide. Southern blot analysis of human genomic DNA using the cloned cDNA as a probe revealed that the human trypsinogen genes constitute a multigene family of more than ten genes

Expression of the human salivary α-amylase gene in yeast and characterization of the secreted protein

Abstract Recombinant plasmids were constructed in which the human salivary α-amylase gene, with or without the N-terminal signal sequence for secretion, was placed under control of the APase (PHO5) promoter of Saccharomyces cerevisiae. In yeast cells transformed with the α-amylase gene having the human signal sequence for secretion, the gene was expressed and the enzyme was secreted into the medium in three different glycosylated forms. The amylase gene without the signal sequence was also expressed in yeast, but the products were neither secreted nor glycosylated. Determination of the N-terminal amino acid (aa) sequence revealed that the 15-aa signal sequence had been cleaved from the secreted enzyme, and that the N-terminal residue, glutamine, had been modified into pyroglutamate, as is commonly observed with the mammalian salivary α-amylase. Thus, the human salivary α-amylase signal sequence for secretion was correctly recognized and processed by the yeast secretory pathway. The C-terminal residue was identified as leucine, which is predicted from the nucleotide sequence data to be located at position 511 in front of the termination codon. Therefore, there is no post-translational processing in formation of the C terminus.

Expression of human salivary α-amylase gene in Saccharomyces cerevisiae and its secretion using the mammalian signal sequence

Abstract A cDNA fragment coding for human salivary α-amylase precursor was joined to the promoter of the Saccharomyces cerevisiae PHO5 gene, and the recombinant gene was inserted into a vector plasmid capable of autonomous replication in yeast. Yeast cells transformed with this recombinant plasmid synthesized about 5 × 10 5 molecules of the enzyme per cell when synthesis was induced by deprivation of inorganic phosphate and released about half of the synthesized enzyme into the medium. The enzyme is stable, and exhibited the same specific activity as α-amylase in human saliva. The amylase-producing yeast grew on starch and produced alcohol.

Production of salivary type α-amylase in human lung cancer

Abstract α-Amylase, which is produced by lung cancer tissue, was studied by cloning cDNAs from a cell line originating from lung cancer that produces amylase. Sequencing studies with this cDNA showed that the expressing gene is of the salivary type. The specific location of the start point of transcription, as revealed by S1 mapping, supported this conclusion.