Extraction of recombinant dog gastric lipase from transgenic corn seed.
Several approaches were examined for extracting the relatively hydrophobic protein recombinant dog gastric lipase (rDGL) expressed in the endosperm of transgenic corn seed. The first approach used minimal processing of the seed before extraction (i.e. simple grinding of whole seed) followed by selective extraction to eliminate 72% of contaminant proteins without compromising rDGL recovery from the meal of whole grain. The second approach added defatting of the whole grain meal to reduce the amount of detergent in the subsequent step for extracting rDGL. The third approach incorporated dry-milling of the corn to recover an endosperm rich fraction, followed by extraction of this fraction. The dry milling strategy was most effective, resulting in recovery of 35 U rDGL/g of corn seed (50 U/g of endosperm) with a specific activity of 9 U/mg compared to 22 U and 3 U/mg for the first strategy and 36 U and 3.7 U/mg for the second. The reductions in host protein contamination and lower detergent levels of the endosperm route should simplify downstream purification steps.
Zhong
Qixin
Q
Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011, USA. qzhong@utk.edu
Gu
Zhengrong
Z
Glatz
Charles E
CE
D000818:Animals; D004285:Dogs; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012639:Seeds / Q000201:enzymology*; D003313:Zea mays / Q000201:enzymology*
D011994:Recombinant Proteins; D008049:Lipase