PURIFICATION AND CHARACTERIZATION OF THERAPEUTIC PROTEIN FROM NERIUM ODORUM

Abstract

The world we live in is full of various harmful microorganisms. Our body temperature and wealth of nutrients provide an ideal home for these organisms to thrive. This has lead to the development of various serious infectious diseases which cause great suffering and burden on human health. Besides, some of the harmful microorganisms also cause serious diseases in plants that affect crops productivity. All animals and plants have developed one or other type of defense mechanism to protect themselves from these pathogenic microorganisms (Tenover, 2006; Sanglard and Odds, 2002). However more often these protections failed, pathogen invades the system and we need to use other means to combat them. Chemotherapy has been the common approach to control these diseases and a large number of chemical agents and antibiotics have been developed or synthesized (Lee, 2005). They have been very effective in controlling the infections but widespread and indiscriminate use of these drugs has resulted in emergence of drug resistance (Lee, 2005) and more recently multidrug resistant pathogenic strains for various human and plant pathogens. The control of infection by these resistant and multidrug resistant strains has been a great challenge mainly because of the existing armory of drug/antibiotics becomes failed to control these infections. In addition, these conventional synthetic drugs and microbial origin antibiotics have side effects (Butler and Buss, 2006). Therefore, there is an urgent need to search for a safer and effective alternative to control and treat these infections. The search for new safer and effective drug which do not cause resistant development is one of the thrust area of biomedical and agriculture sciences (Butler and Buss, 2006). Attempts have been made to isolate therapeutic agents from diverse group of organisms including microbes (Chernin et al., 1997), fungi (Hao et ah, 1999; Hao et ah, 2000), animals (Iijima et ai, 1993) and plants (Lee et ah, 2007; Huynh et al, 1992). Because of the highly diversified flora and their diversified phytochemical constituents, plants are thought to be a better choice for search of new therapeutic agents (Butler and Buss, 2006). Among the numerous compounds, bioactive proteins have been found of prime importance because they often have very specific interactions with a macromolecular target in the body. On the basis of their beneficial health effects they may be classified as antimicrobial, antioxidative, antidiabetic, antithrombotic, antihypertensive, anticancer and immunomodulatory (Liu et al, 2007; Ghosh and Maiti, 2007; Hsu et al, 1997; Fisher and Yang, 2002; Sheu et al, 2004). Among the various biologically active proteins identified, the present discussion is mainly inclined towards antimicrobial and immunomodulatory proteins. A large number of antimicrobial proteins/peptides (AMP) have been isolated from various plant sources (Wang and Ng, 2002; Hao et al, 1999; Hao et al, 2000; Lee et al, 2007; Huynh et al, 1992; Iijima et al, 1993) and extensive research has been done on their structure and mode of action (Brogden, 2005; Otvos Jr et al, 2005). The best known plant's antimicrobial proteins comprising thaumatin-like proteins (Chu and Ng, 2003; Graham et al, 1992; Huynh et al, 1992; Pressey, 1997; Vu and Huyhn, 1994; Wurms and Greenwood, 1999; Ye and Ng, 2000), chitinases (Van Damme et al, 1993; Vergauwen, 1993), chitinase-like proteins (Lam et al, 2000; Ye et al, 2000), chitin binding proteins (Huang et al, 2000; Van Den Bergh et al, 2004), defensins, defensin-like proteins (Ngai and Ng, 2004; Wong and Ng, 2003; Wong and Ng, 2005), miraculin-like proteins (Ye and Ng, 2000), embryoabundant proteins (Wang and Ng, 2000), ribosome inactivating proteins (Leah et al, 1991), lipid transfer protein-like proteins (Cammue et al, 1995; Wang et al, 2004), protease inhibitors (Chen et al., 1998; Chilosi et al, 2000; Joshi et al, 1998; Loritoet et al, 1994) and new proteins/peptides (Chu et al, 2005; Ng and Wang, 2000; Xia and Ng, 2005). A 42 kDa chitin-binding proline-rich protein (PRP) from French bean has been characterized through its involvement in plant-pathogen interactions (Bindschedlera et al, 2006). An antifungal protein was purified from Alocasia esculenta rhizomes showing activity toward Botrytis cinerea and resembles pisavin, the miraculin-like anti-fungal protein from sugar snap in N-terminal sequence (Wang and Ng, 2003). Ginkbilobin, a novel antifungal protein from Ginkgo biloba seeds was isolated with sequence similarity to embryo-abundant protein (Wang and Ng, 2000). An antibacterial and antifungal protein was also isolated from Indigofera oblongifolia leaves (Dahot, 1999) with activity toward Escherichia coli, Klebsiella aerogenes, K. pneumoniae, Staphylococcus aureus, Bacillus subtilis, Aspergillus fumigatus, A. niger and A.flayus. A 21 kDa thaumatin-like protein has been isolated from green kiwi fruits, and exhibits HIV-1 reverse transcriptase inhibitory activity and antifungal activity against a number of fungi (Lixin and Ng, 2004). There are several excellent review articles discussing various aspects ofAMP (Ghosh and Maiti, 2007; Wasser, 2002; Winder et al, 1998; Wong and Ng, 2005). The immune system plays an important role in the protection of the body from disease. It protects against not only those diseases which result from an attack by bacteria, virus, and other pathogens, but also cancer, as well as disease states which result from immune imbalance; opportunistic infections, or autoimmune disorders. Modulation of the immune system through pharmaceutically induced stimulation or suppression offers an important approach to the control of diseases. Therefore, search for the novel immunomodulatory agents have been one of the important area of biomedical research. A number of naturally occurring proteins from plants with in vitro and/or in vivo immunomodulatory activities have been reported from various plants (Pugh et al, 2001; Wasser, 2002; Hsu et al, 2004; Ou et al, 2005; Liu et al, 2007; Hsu et al, 1997; Vivanco 1997). Some of the known immunomodulatory proteins are 13 kDa LZ-8 protein from Ganoderma lucidum, 15 kDa Fip-vvo protein from Volvariella volvacea, 31.5 kDa and 205 kDa lectins from Agrocybe cylindracea, and Cteropharyngodon idellus, 13.8 kDa napin-like polypeptide from Chinese cabbage seed, arabinogalactan (AG) from Nerium oleander and arabinogalactan-proteins (AGPs) from Echinacea purpure. In recent few years, there has been sudden increase in bacterial and fungal infections caused by opportunistic and drug resistant pathogens particularly in immuno compromised host and patients who has gone for transplant surgery. A therapeutic molecule with inherent properties of both antimicrobial as well as immunomodulatory activity will be more effective in controlling and treating the antimicrobial infections in general and in immunocompromised hosts in particular. As such novel molecules will have dual action, first by killing the pathogen and second by boosting the immunesystem of the host, but finding such molecules is difficult task. However, the observations that several antimicrobial proteins/peptides besides their antimicrobial action also found to have immunomodulatory effects provide strong basis that such proteins do exist in nature. Keeping this fact in mind, an attempt has been made in the present study to search for such a novel proteins from Nerium odorum a well known medicinal plant. Nerium odorum Syn. N. indicum (Mill) common name oleander is a widely distributed evergreen plant belonging to the family of Apocynaceae. It is a large, erect, stout shrub with milky juice, linear lanceolate, thickly coriaceous, acuminate leaves, with faintly sweet-scented white or red colored flowers. N odorum is well known for its medicinal uses in immune deficient diseases including cancer and AIDS. Whole plant is said to have anticancer properties (Manna et al, 2000). An immunologically active acidic pectic polysaccharide has been isolated and characterized from the leaves (Mueller et al, 1991). Oleandrin, a polyphenols cardiac glycoside from leaves possess anti inflammatory and tumor cell growth-inhibitory effects (Stenkvist, 1999). The fresh juice of leaves is dropped into the eyes for inducing lachrymation in ophthalmia (Hussain, and Gorsi, 2004). A decoction of the leaves has been applied externally in the treatment of scabies (Kirtikar et al, 1975). Paste of the root is used as an external application in hemorrhoids, and ulcerations (Chopra et al, 1956). The root of this plant is a good tonic for chronic pain in the abdomen and joints and used as an antidote to snake-venom (Hussain and Gorsi, 2004). Oil extracted from the root bark is used in leprosy and other diseases of a scaly nature. However, there is no report on the study of therapeutic proteins from this plant. The present work was emphasized to search a protein with potential antimicrobial and immunomodulatory activity from TV. odorum. This study was undertaken with the following objectives: 1. Isolation and purification of antimicrobial protein from the leaves ofTV. odorum. 2. Characterization of purified protein by different biochemical methods (SDS PAGE and HPLC) and biophysical methods (N-terminal sequencing, and mass spectrometric technique). 3. Evaluation of antimicrobial potential of purified protein using standard methods (disk diffusion and broth dilution assays, SEM and TEM). 4. Evaluation of immunomodulatory activity of purified protein in in vitro and in vivo system. 5. Evaluation of cytotoxicity of purified protein.