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Introduction: Proteases are the most important industrial enzymes. Bacillus bacteria are commonly used to produce these enzymes. The aim of this study was cloning, sequencing, expression and bioinformatics study of aprX serine protease gene extracted from Bacillus licheniformis.
Materials and Methods: In this study, after extraction of bacterial DNA, aprX serine protease genes was isolated from Bacillus licheniformis and cloned into pTG19-T vector and then subcloned in pET28a vector. The molecular structure, its biochemical and phylogenetic properties were investigated and three-dimensional structure of the cloned enzyme was predicted. For the induction of gene expression and protein production of the recombinant serine protease IPTG was used at different concentrations, different temperatures and different time periods. Confirmation of aprX gene expression was performed by SDS-PAGE and dot blot analysis. Then, the activity of recombinant protease enzyme was measured at different temperatures and pHs.
Results: Cloning was confirmed by sequencing. Based on the results of phylogenetic studies, the obtained protein sequence showed a high similarity to the sequences of other Bacillus species. After evaluating the drawn models, it was found that the models provided by RAPTROX and I-TASSER software were desirable models for predicting the three dimentional structure of this protease. The recombinant protein production was successfully induced by IPTG induction in the host containing the plasmid pET28a-aprX. The highest expression values were obtained at 25 ° C for 20 hours with 0/5 mM IPTG. Also, the recombinant protein produced showed the highest activity at 50 ° C and pH 8.

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Objective: The incidence of breast cancer is approximately one million which makes this cancer one of the most common among women worldwide. Breast cancer comprises 7% of the total death rate caused by cancers. Several strategies that use tumor-associated antigen (TAA) vaccination and early detection of breast cancer are clinically being developed. Breast cancer is caused by increased over expression of certain genes. HER-2 is a tyrosine kinase receptor in the epidermal growth factor family. The role of HER-2 in breast cancer has been extensively studied. HER-2 is found in 25%-30% of breast cancer patients. Herceptin, a human antibody, is used as a therapeutic target for HER-2. The purpose of this study is to produce recombinant protein HER-2 for early detection of breast cancer cells. Methods: We used specific primers to amplify the HER-2 gene. The amplified gene was cloned into pET28a as an expression vector. Cloning was confirmed by restriction analysis and sequencing. Expression was induced using IPTG and the recombinant protein was analyzed by SDS-PAGE. Results: Cloning of the HER-2 gene was confirmed by enzyme digestion and sequencing. The gene was expressed in E.coli BL21 DE3. The pET-28a vector which contained the HER-2 gene showed a high level of expression. The recombinant protein was confirmed by Western blot analysis. Conclusions: A portion of the HER-2 gene was expressed as a recombinant in E.coli. This could be a good diagnostic test for breast cancer.