HomePhilippine Journal of Veterinary Medicinevol. 41 no. 2 (2004)

Yields of Recombinant Carboxy-Terminal End of the Botulinum Neurotoxin Types C and D Heavy Chain Expressed in Escherichia coli DH5a Cells using Three Extraction Procedures

Mary Joy N. Gordoncillo | Lynn A. Woodward | Robert G. Hirst

Discipline: Veterinary Medicine

 

Abstract:

Plasmid constructs pC50 and pD50 containing the region encoding for the carboxyterminal end of the heavy chain (He) of Clostridium botulinum types C and D, respectively, were cloned into Escherichia coli DH5a cells for expression. Because high levels of expression in prokaryotic systems such as this is limited, maxim urn recovery of the recombinant proteins from the culture is warranted. To attain this, three different extraction procedures were compared: extraction using 8M urea alone, using French press, and by combination of 8M urea and sonication of transformed cells. For pC50 and pD50, extraction method using 8M urea gave 0.48 and 0 .04 mg/l of culture, respectively, while the use of French press yielded 0.86 and 1.27 mg/l of culture, respectively. Extraction using the combination of.8M urea and sonication of cells, gave the highest yield of 10.8 mg/l of culture for pC50 and 9.9 mg/l of culture for pD50, and is therefore recommended for this purpose.