Evidence Based Comparative Studies on free and Immobilized Therapeutic Protein

Rahamat Unissa, Ashwini Kumari Chauhan , Asmathpet Malleshwari, Narsareddy Vinitha, Karra Haarika, Chundru Sri Aneesha, Datti Navya Teja Sree, Mounika Sangishetty and Ishrath Begum

Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Malla Reddy College of Pharmacy, Maisammaguda, Dhulapally, Secunderabad, Osmania University, Telangana, India.

Corresponding Author E-mail: srunissa@gmail.com

DOI : http://dx.doi.org/10.13005/bbra/2451

ABSTRACT: The aims of  the present work were to carry out comparative studies on free and immobilized l-arginine deiminase obtained from marine Vibrio alginolyticus 1374. Therapeutic proteins of microbial origin though possess excellent pharmacological activities gets quickly broken down into amino acids by the action of proteases in the body. Hence to protect its activity and integrity the enzyme was attached with a compatible polymer such as PEG and was further evaluated for the presence of the stability and activity. Enzyme production was carried out by culturing Vibrio sp. under optimal conditions and was purified by standard chromatographical techniques. The purified enzyme thus obtained was immobilized using different concentrations of PEG20k and purified. The Pegylated enzyme was kinetically characterized and evaluated for anti-proliferating activity against four cancer cell lines. mPEG-Succinimidyl Succinate, MW 20,000 coupled with ADI with very high affinity under mild conditions. The pegylated ADI had 3.1 PEG chains of 20k length which not only protected the enzyme from degradation but also increased plasma t ½ and prevented immunogenicity. It showed approximate molecular weight of about 112 k Da. The K_m and V_max values of PEGylated ADI were found to be 2.94 ± 0.13 mM and 129.87 ± 1.24 U/ml/min respectively. Though there was a slight change in K_m and V_max values, it still retained its activity against four cancer cell lines with slight decrease in the activity compared to free enzyme. L-Arginine deiminase of  Vibrio sp. remained effective even after pegylation. Hence it can be a promising candidate in treatment of l-arginine auxotrophic cancers. In vivo studies must be carried out to develop the enzyme in the form of chemotherapeutic drug.

KEYWORDS: A375-C6; argininosuccinate synthetase;  clone E6-1cell lines HCT-113 and jurkat;MCF-7;  ornithine carbamoyl transferase; Vibrio alginolyticus 1374;

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