Cloning And Expression Of Protease Genes From Keratin-Degrading Strain Streptomyces Fradiae Var.K-11

Keratin is a structural protein that can be found extensively in hairs, squamas, feathers, hoofs and horns of animals. It is chemically stable, insoluble in water and can't be degraded by ordinary proteases, such as trypsin, pepsin and papain. Keratinases purified from microbes have strong activities, which can hydrolate many obstinate fibrins, such as keratin, elastin and collagen. So they have extensive application prospects.This reseach reported the optimal culture condtions for efficient production of proteases with strong activity in Streptomyces fradiae var. k11. Six secreted proteases were identified from supernatant of k11 culture medium by isoenzyme electrophoresis analysis. This result made the first step towards the revelation of secreted protease system in k11.Expression patterns of the six protease genes in feather medium and Gaos medium were examined by RT-PCR analysis. The results showed that the six protease genes were expressed constitutively.Two of the six proteases were purified successfully and partially sequenced.Genomic DNA library of Streptomyces fradiae var. kl 1 was constructed. Genes encoding the six proteases were cloned from the library using the combination of several gene cloning strategies, and four genes, sfp1, sfp2, sfp3 and sfap, have been registered in EMBL (Accession number: AJ784159, AJ784940, AJ810091, and AJ781828). The sfp1, sfp3 and sfap have less than 80% homologies with DNA sequences of proteases ever reported, and the protein homologies are all less than 72%. So they are three novel protease genes.The encoded amino acid sequence of another k11 originated protease gene sjpl shared 99.5% homology with a reported protease Sfase-2, so they could be treated as one protease. Former researches indicate that this protease has strong keratinase activity and good application potential. Only part (492bp) of its coding sequence (576bp, encoding mature protein) has been reported. Its zymogen ,signal peptide sequence and gene flanking sequence was all unknown before, and gene function was also not verified. "Pro" part of the protease may play an important role in enzyme function. In this study, the protease was purified and partially sequenced, partial gene sequence was cloned from genomic DNA library constructed, and then the complete gene including DNA sequences encoding signal peptide and "pro" sequence is cloned successfully.Constutive and sucrose inducible Bacillus subtilis expression systems were also established for further utilization of genes identified in this study.In E. coli strain BL21, sfp1 and sfp2 were expressed and functionally verified. Both mature protease gene and zymogen gene of sfp2 were expressed successfully in E. coli and B. subtilis, respectively, and it had been proved that pro-peptide played important roles in proper folding and...