Assessment of the Cucumber Mosaic Virus Coat Protein by Expression Evaluation in a Genetically Modified Pepper and Escherichia Coli Bl21

Abstract

A genetically modified (GM) pepper containing the gene for the cucumber mosaic virus (CMV) coat protein (CP) was investigated to assess the proteolytic behavior and simulated digestion in the stomach and small intestine. CMV‐CP expressed in GM pepper and in Escherichia coli exhibited the molecular mass of about 25 kDa on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. The three internal peptides of CMV‐CP in E. coli BL21 (VLSQQLSR, LLLPDSVTEYDKK and DDALETDELVLHVDIEHQR) were detected by electrospray ionization mass spectrometry/mass spectrometry, and seven peptides (VLSQQLSR, PTINHPTFVGSER, LLLPDSVTEYDKK, FDSTVWVTVRK, LLYDLSAMRA, RKYAVLVYSK and VLHVDIEHQR) of CMV‐CP in GM pepper were verified by the analysis. CMV‐CP expressed in GM pepper was completely degraded in simulated gastric and intestinal fluids, and the immunoreaction of the CMV‐CP was rapidly eliminated by the Western blot assays. PRACTICAL APPLICATIONS A variety of cucumber mosaic virus (CMV‐P0)‐resistant peppers was developed through a breeding program, and commercial cultivars are available. However, a new pathotype virus (CMV‐P1), which can infect CMV‐P0‐resistant peppers, is appeared recently, and this virus causes a heavy loss of productivity. Recently developed new transgenic pepper line is highly tolerable to CMV‐P0 and CMV‐P1. Sequence analysis of the CMV‐coat protein (CMV‐CP) expressed in the new transgenic pepper line and in an Escherichia coli expression system will provide structural information on expressed protein. In addition, in vitro digestibility of the expressed CMV‐CP from transgenic pepper with simulated gastric fluid and simulated intestinal fluid make it possible to understand its proteolytic behavior with simulated digestion fluids.

Improved detection of Cucumber mosaic virus coat protein (CMV-CP) in genetically modified pepper (Capsicum annuum) using a polyclonal antibody to a synthetic CP peptide

“…Membranes were stained using nitroblue tetrazolium (Promega, Madison, WI, USA) and 5-bromo-4-chloro-3-indoyl phosphate (Promega, Madison, WI, USA) as substrates in alkaline phosphatase buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl 2 ) for less than 10 min, and the reaction was stopped when the desired color density was obtained. Soluble proteins from GM and non-GM peppers cultivated in Korea University fields were extracted as described previously (Bang et al, 2011). Frozen peppers (20 g) were homogenized in a pre-chilled blender with chilled 0.05 M potassium phosphate buffer, pH 8.0, containing 1.0 mM ethylenediaminetetraacetic acid (EDTA), 1.0 mM phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (SigmaAldrich, St. Louis, MO, USA) (buffer A).…”