www.IndianJournals.com Members Copy, Not for Commercial Sale Downloaded From IP - 117.240.224.35 on dated 22-Jun-2010 Red Fluorescent Protein from Silkworm 63 Purification and Partial Characterization of Red Fluorescent Protein from Silkworm, Bombyx mori GS SURESH 1 , LN SRIKAR, PETER ANITHA, L SHANTHALA 2 and M A SHANKAR 3 Department of Biotechnology, 2 Department of Crop Physiology, 3 Dry Farm, University of Agricultural Sciences, Gandhi Krishi Vignyan Kendra, Bangalore-560065, India 1 Indian Agricultural Research Institute, New Delhi-110 012, India A red fluorescent protein (RFP) from silkworm gut juice was isolated and purified by a combination of ammonium sulfate fractionation and gel filtration chromatography. The elution profile of gel filtration chromatography on Sephadex G-100 showed three peaks at 280 nm, of this only one peak showed high absorbance both at 280 nm and 605 nm, which is characteristic of RFP. Fractions constituting the major peak showed red fluorescence under UV light. Electrophoretic analysis on SDS-PAGE revealed that RFP is a single protein band having molecular weight of 66 kDa. Antiviral activity of RFP against nuclear polyhedrosis virus (NPV) through bioassay studies revealed that larvae treated with NPV + RFP reduces the percent mortality to 44% compared tothe larvae treated with NPV + BSA (reference protein) having 84% mortality. Immuno characterization of purified RFP showed that RFP content in the digestive juice of silkworm depends on the quality of mulberry leaves. Increased application of nitrogen and potassium to mulberry increased the RFP content in silkworm. Key words: Red fluorescent protein, antiviral activity, nuclear polyhedrosis virus (NPV) Indian J Agric Biochem 20(2), 63-68, 2007 *Corresponding Author : Email- sureshgiriyapur@gmail.com The defence arsenal of silkworm contains certain immune responses against pathogens.The red fluorescent protein (RFP) is one of the factors, the biosynthesis of which requires chlorophyll from mulberry. The RFP combines with larval gut protein in the presence of light (1, 2). It was found that RFP and an alkaline protease in silkworm gut juice showed antiviral activity against Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) (3, 4). Unchida et al (3) isolated and purified the RFP from the digestive juice of silkworm larvae. The SDS-polyacrylamide gel electrophoresis and the ultra-centrifugal analysis showed that the purified substance was a homogenous simple protein. The molecular weight of the purified protein was found to be 27- 28 kDa and the sedimentation coefficient was 2.61S, and also had an additional activity of inactivating NPV of Bombyx mori in-vitro. Hou and Chiu (5) suggested that RFP contains more than one midgut protein and the antiviral activity of purified RFP in silkworm gut juice is attributable to phospholipase C (PLC) activity. Funakoshi and Aizawa (4) described the characteristics of an antiviral substance in the gut juice active against BmNPV by gel filtration on Sephadex G-75. The separated fraction showed strong antiviral activity and a high protease activity. Sethuraman et al . (6) purified the RFP from silkworm gut juice by Sephadex G-100 column chromatography. The elution profile showed three peaks at 280 nm. However, only one peak showed high absorbance both at 280 and 605 nm, which is characteristic of RFP. Electrophoretic separation of purified RFP showed only a single protein of 65 kDa and the antiviral nature of the isolated protein was confirmed against BmNPV. Datta et al. (7) purified and characterized the antiviral protein in the gut juice of silkworm (Bombyx mori L.) The present study was carried out to isolate, purify and quantify RFP when silkworm larvae was fed with mulberry leaves grown under different levels of N, K and Zn and relate this to the disease tolerance. Materials and Methods Protein molecular weight markers were obtained from Bangalore Genie Company. TEMED was from Koch-light laboratories Ltd., England, Sephadex G-100, Tween 20 and Freund’s complete and incomplete adjuvants were optained from Sigma Chemical Co. USA. Silkworms of the breed Kolar Gold (PM X CSR 2 ) were reared at MRS, Hebbal, UAS, Bangalore. Fifth instar larvae were used for extraction of digestive juice from the midgut. Purification Procedure: The procedure adopted for the purification of RFP from digestive juice of Bombyx mori was as described by Uchida et al. (3) and as modified by Sethuraman et al. (6). All procedures were carried out at temperature below 5 o C. The digestive juice was collected