Cloning and Expression of Trypsin-Like Enzyme from Streptomyces fradiae for Comparative Analysis of Functional Regions of Streptomyces and Amammalian Trypsins

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The trypsin-like enzyme from Streptomyces fradiae (SFT) is one of the extracellular proteases secreted by gram-positive bacteria. Since the primary structure of SFT is still unknown, a gene encoding SFT was cloned from a S. fradiae genomic library using an amplifed polymerase chain reaction product within the SFT gene as a probe. The nucleotide sequence of the cloned fragment revealed that the gene encoded an open reading frame with 259 amino acids. The 38 N-terminal amino acids resemble a typical prokaryotic signal peptide, but the predicted signal sequence cleavage site suggests the existence of a very short, four-amino-acid prosequence. The mature SFT consists of 221 amino acids with a molecular weight of 22900. SFT was expressed in Streptomyces invidans 1326 using pIJ702 as a vector, and the secreted protein was purified from culture supernatant by soybean trypsin inhibitor-affinity chromatography. The N-terminal amino acid sequence and molecular weight of the protein were identical to those of natural SFT, indicating correct processing by S. lividans 1326. Also, the amino acid composition of the recombinant SFT agreed with that of natural SFT and that deduced from the nucleotide sequence. Comparison of the amino acid sequence of SFT and other trypsins of microbial and mammalian origins revealed that SFT exhibits 85% identity to Streptomyces griseus trypsin (SGT), but low identity to Saccharopolyspora erythraea trypsin (SET) (38%) and to bovine trypsin (35%). The sequence alignment shows that the catalytic triad, the substrate-binding site and six cysteine residues are highly conserved. We found amino acid substitutions between SFT and SGT in regions involved in substrate specificity and sequence differences between Streptomyces and mammalian trypsins in substrate-binding regions.
社団法人日本生物工学会の論文
1995-11-25

Cloning and Expression of Trypsin-Like Enzyme from Streptomyces fradiae for Comparative Analysis of Functional Regions of Streptomyces and Amammalian Trypsins

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