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:: Volume 2, Issue 7 (9-2012) ::
NCMBJ 2012, 2(7): 27-34 Back to browse issues page
Cloning, Gene Expression, Purification and Characterization of Elastase from Pseudomonas Aeruginosa
Somayeh Ehtesham Ehtesham , S. Mohsen Asghari , Afsaneh Sadre momtaz
Department of Biology, Faculty of Sciences, Islamic Azad University, Parand Branch,Tehran-Iran , smy.ehtesham@yahoo.com
Abstract:   (22127 Views)

Aim and Background. Pseudomonas Aeruginosa Elastase is an important enzyme in basic studies on Zn-metalloproteases and is also considered as a virulence factor of the bacterium. In the present study, the gene encoding Pseudomonas aeruginosa elastase (PAE) was extracted and clone, Following expression and purification, its biochemical properties including optimal temperature and pH, and also enzymatic activity in the presence of glycerol, dimethylformamide (DMF), methanol, ethanol, ethylene glycol and isopropanol were investigated.

Materials and Methods. Bacterial strain was verified by biochemical tests including citrate, SIM, MR-VP and TSI. Genomic DNA was extracted using gene extraction kit and elastase gene was amplified by PCR using two specific primers. Cloning was carried out within pET21a (+) vector with HindIII and NdeI restriction sites and transformation accomplished by chemical method. The cells were cultured under the given optimized conditions, in LB medium with 1mM IPTG.

Results. Following the strain verification by biochemical tests, genomic DNA was extracted and PAE encoding gene was isolated using specific primers. Elastase gene, which is encoded as pre-proelastase, was cloned within pET21 a (+) vector and transformed into E.coli strain BL21. Nucleotide sequence analysis shows an open reading frame (ORF) of 1497 nucleotides corresponding to 497 amino acid residues. Following the induction by IPTG, the active enzyme was detected in the soluble fraction. Consequently, the enzyme was purified using affinity chromatography. Enzyme properties such as optimal temperature and pH, irreversible thermo inactivation and organic solvent activity were investigated. Our results clearly indicate that the above mentioned enzyme possesses a noticeable activity and stability within organic media.

Conclusion. Elastase from Pseudomonas aeruginosa strain PTCC1430 shows only one amino acid substitution in the signal sequence in comparison with that of strain PAO1 whose crystal structure is solved. Biochemical properties reveal that elastase is a highly stable enzyme in organic solvents.

Keywords: Pseudomonas Aeruginosa Elastase, cloning, Expression, Purification, Characterization.
Full-Text [DOC 31 kb]   (7261 Downloads)    
Type of Study: Research Article | Subject: Biochemistry
Received: 2012/09/15
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Ehtesham S E, Asghari S M, sadre momtaz A. Cloning, Gene Expression, Purification and Characterization of Elastase from Pseudomonas Aeruginosa. NCMBJ. 2012; 2 (7) :27-34
URL: http://ncmbjpiau.ir/article-1-206-en.html


Volume 2, Issue 7 (9-2012) Back to browse issues page
مجله تازه های بیوتکنولوژی سلولی - مولکولی New Cellular and Molecular Biotechnology Journal
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