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

Alan C. Antony, Vimi Louis, Darsana Dilip, K. Anita Cherian

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.


Keywords


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

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A KAU publication [CODEN: JTAGEI; ISSN 0971-636X; eISSN 0973-5399]