Heterologous expression of the alpha-amylase inhibitor gene cloned from an amplified genomic sequence of Streptomyces tendae.
The coding region for a secreted proteinaceous inhibitor of the human alpha-amylase (tendamistat; HOE 467) was identified by using a synthetic oligonucleotide probe. The gene is part of a 37-kilobase amplified genomic sequence found in an overproducing mutant of Streptomyces tendae. After subcloning, sequence analysis revealed an open reading frame of 312 base pairs preceded by a putative ribosome-binding site. The reading frame is 30 codons longer than necessary for the mature protein. This sequence coded for an amino-terminal extension of tendamistat and shows typical features of a signal peptide. After being cloned into Streptomyces vector plasmids and transformed to the heterologous host, Streptomyces lividans TK24, the gene was expressed, and the alpha-amylase inhibitor was correctly processed and secreted into the culture medium. The amount of secreted protein was dependent on the gene dosage and on the promoter arrangement.
Koller
K P
KP
Hoechst AG, Frankfurt am Main, Federal Republic of Germany.
Riess
G
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D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004274:DNA, Recombinant / Q000378:metabolism; D005784:Gene Amplification; D005796:Genes; D005798:Genes, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010455:Peptides / Q000235:genetics*; D015183:Restriction Mapping; D013302:Streptomyces / Q000235:genetics*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors*
D003062:Codon; D004274:DNA, Recombinant; D015345:Oligonucleotide Probes; D010455:Peptides; D000516:alpha-Amylases; C030526:tendamistate