Purification of recombinant Cucumber mosaic virus (banana isolate) coat protein by sucrose density gradient ultra-centrifugation

Purification of recombinant Cucumber mosaic virus (banana isolate) coat protein by sucrose density gradient ultra-centrifugation

Authors

  • Alan C. Antony College of Agriculture, Kerala Agricultural University, Thrissur 680 656, Kerala, India
  • Vimi Louis Banana Research Station, Kerala Agricultural University, Thrissur 680 652, Kerala, India
  • Darsana Dilip College of Agriculture, Kerala Agricultural University, Thrissur 680 656, Kerala, India
  • K. Anita Cherian College of Agriculture, Kerala Agricultural University, Thrissur 680 656, Kerala, India

Keywords:

Cucumber mosaic virus, Sucrose density gradient ultra-centrifugation, Buffer, Recombinant protein

Abstract

Cucumber mosaic virus (CMV) infected banana leaf samples were collected based on characteristic symptoms and screened by direct antigen coating immunosorbent assay and reverse transcriptase polymerase chain reaction. The expression clone of CMV coat protein was prepared using the expression vector pRSETC and the host Escherichia coli BL21 (DE3)pLysS. Then, E. coli cells were induced by Isopropyl â-D-1- thiogalacto pyranoside (IPTG) for expression of 25 kDa recombinant CMV coat protein. After induction, the E. coli cells were sonicated and the coat protein was ultra-pelleted. Sucrose density gradient (10-40%) was prepared and the coat protein ultra- pellet which was dissolved in Tris-NaCl buffer of pH 8.0 was subjected to sucrose density gradient ultra-centrifugation at 26,000 rpm for 3 h at 4°C for purification. The SDS-PAGE gel profile of sucrose fractions indicated more recombinant CP in 10- 20 per cent sucrose fractions. Along with the recombinant CP, contaminant (host derived) proteins were also observed. When the coat protein ultra-pellet was dissolved in SAT buffer of pH 5.5 and subjected to sucrose gradient ultracentrifugation as above, the 25 kDa coat protein was absent in sucrose fractions and was observed in cell pellet which indicated the insolubility of the protein in the buffer.

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Published

18-01-2021

How to Cite

Antony, A. C., Louis, V., Dilip, D., & Anita Cherian, K. (2021). Purification of recombinant Cucumber mosaic virus (banana isolate) coat protein by sucrose density gradient ultra-centrifugation. Journal of Tropical Agriculture, 58(2). Retrieved from https://jtropag.kau.in/index.php/ojs2/article/view/1004

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