ISSN: 2278-778X Research Article www.ijbio.com International Journal of Bioassays (IJB) 196 EXPRESSION AND PURIFICATION OF LECTIN A FROM PSEUDOMONAS AERUGINOSA IN E. COLI B Veeresh, G Adarsh, T Ramesh, M Ramesh* Department of Biotechnology, JNTU Kakinada-UCEV, Andhra Pradesh, India *Corresponding Author: Dr. Ramesh Malothu, Assistant Professor and Head, Department of Biotechnology, JNTU, Kakinada-UCEV, Vizianagaram, Andhra Pradesh, India. Received for publication: October 11, 2012; Accepted: October 28, 2012. Abstract: Pseudomonas aeruginosa is a pathogenic micro organism which infects cystic fibrosis patients. The mode of infection is by attaching to the host cell using lectin proteins present in cell wall of Pseudomonas aeruginosa. The genomic DNA has been isolated from Pseudomonas aeruginosa. The Lec A gene coding for lectin protein has been isolated from genomic DNA of Pseudomonas aeruginosa using specific primers by specific gene amplification using PCR. The isolated Lec A gene is cloned by T/A cloning. The Lec A gene is inserted into expression vector pET-32a. The recombinant vector is transformed into E. coli BL-21. The protein is over expressed by adding inducer IPTG. The expressed protein is purified by using affinity chromatography and confirmed by SDS PAGE. Keywords: Lec A, Pseudomonas aeruginosa, PCR, SDS PAGE, Affinity chromatography. INTRODUCTION Pseudomonas aeruginosa is an opportunistic pathogen, which is involved in acute infections, as well as chronic infections, especially in cystic fibrosis patients (King EO et al.,). The therapeutic options for these infections remain limited because this pathogen exhibits increasing resistance to many antibiotics (D'Argenio DA et al.,). Currently, antibiotic research and development are at an all-time low, and few new anti Pseudomonal compounds are in the pipeline. Therefore, there is a need for therapeutic approaches other than antibiotics. For P. aeruginosa, as for other pathogenic microorganisms, the ability to adhere to host tissues is essential for initiating infection. Adhesion is often mediated by host cell surface glycoconjugates, which are a specific target for bacterial receptors (Williams HD et al.,). Such oligosaccharide-mediated bacterium- cell recognition and adhesion have been shown to be crucial in the early steps of P. aeruginosa pathogenesis. P. aeruginosa adhesion is mediated by a glycanic recognition pattern involving several adhesins, including lectins. Only a limited number of the carbohydrate-binding proteins of P. aeruginosa have been studied, and their role in recognition and adhesion is far from being elucidated. Two soluble lectins, LecA (PA-IL) and LecB (PA-IIL), specifically binding galactose and fucose, respectively, were initially identified and characterized in the cytoplasm of P. aeruginosa (Vander Wauven C et al.,). However, large quantities of both of these lectins are present on the outer membrane of the bacteria, suggesting that lectins may play a role in adhesion (Hachem RY et al.,). These two lectins, which are produced by the bacteria, are also associated with virulence factors and regulated by both quorum sensing and the alternative sigma factor RpoS (Forestier C et al.,), suggesting that they are also parts of the numerous systems involved in P. aeruginosa virulence. The P. aeruginosa Lec A gene can be expressed in E.coli to produce in bulk amount and then it can be used in the preparation of vaccine to treat P. aeruginosa infections. MATERIALS AND METHODS Isolation of genomic DNA from Pseudomonas aeruginosa by potassium acetate method: The genomic DNA from Pseudomonas aeruginosa has been isolated using the following protocol. Overnight grown culture of Pseudomonas aeruginosa in 2% LB broth at 37°C is harvested till good pellet is formed by centrifuging at 5000 rpm for 5 min in a 1.5ml microfuge tube. The pellet is re suspended in 575μl of 1X TE buffer. 150μl of 10% SDS is added, mixed well and incubated for 1hour at 37°C. 150μl of 5M potassium acetate is added, mixed well by inverting and it is incubated on ice for 15 min. It is centrifuged at 10,000 rpm for 10 min, and the supernatant was transferred in to a fresh vial. 1μl of RNase A is added to it and incubated for 30 min at 37°C. 0.6ml of isopropanol is added to it, mixed gently and allowed it precipitate at - 20°C for 15-30 min. It was centrifuged at 12,000 rpm for 8 min at 4°C and the supernatant is discarded. 400μl of 70% ethanol is added and it was centrifuged at 12,000 rpm for 5 min at 4°C. The supernatant is discarded; the pellet is dried by inverting the vial on the tissue paper towel. The pellet is dissolved in 40μl of TE buffer and stored at 4°C. DNA is electrophoresed on 0.8% agarose gel to observe the bands.