Isolation, Molecular Cloning, Expression and Bioinformatics Evaluation of aprX Serine Protease Gene Extracted from Bacillus licheniformis

Aghaei Jeshvaghani, Zahra; Hosseini, Ramin

Isolation, Molecular Cloning, Expression and Bioinformatics Evaluation of aprX Serine Protease Gene Extracted from Bacillus licheniformis

Document Type : Original Research

Authors

zahra aghaei jeshvaghani ¹

Ramin Hosseini ²

¹ PhD student, Department of Biotechnology, Faculty of Agriculture and Natural Resources, Imam Khomeini International University

² Associate Professor, Department of Biotechnology, Faculty of Agriculture and Natural Resources, Imam Khomeini International University

Abstract

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.

Keywords

Expression optimization

Recombinant Protein Expression

Sequencing

Dot blotting

Protein modeling

Molecular cloning

20.1001.1.23222115.1400.13.1.5.4

Subjects

Molecular biotechnology

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