Recovery of Recombinant Dog Gastric Lipase from Corn Endosperm Extract

Abstract

Two separation methods, aqueous two‐phase (ATP) partitioning and cation‐exchange chromatography, were compared as alternative methods for the recovery of recombinant dog gastric lipase (r‐DGL) from extracts of transgenic corn endosperm. r‐DGL is a hydrophobic, acid‐stable protein targeted for stable expression in endosperm. Polyethylene glycol (PEG) ‐ salt ATP system parameters of PEG molecular weight, phase‐forming salt, NaCl addition, Triton X‐100 concentration and phase ratio were adjusted to achieve favorable partitioning. The purification factor and yield of r‐DGL in the bottom phase of a PEG 3350 (14.2%)‐Na₂SO₄ (8.5%)‐NaCl (0.5%)‐Triton X‐100 (2 mM) system at pH 4 were 1.5 and 80%, respectively. A higher purification factor of 2.3 and nearly 100% yield of r‐DGL was obtained in the top phase of a PEG 3350 (9.4%)‐phosphate (14.3%)‐NaCl(1.5%)‐Triton X‐100 (2 mM) system at pH 4.0. The yield, purification factor, and concentration factor were 90%, 7.7, and 3.6, respectively, for the alternative of cation‐exchange on CM‐Sepharose. Countercurrent ATP partitioning with 3–7 stages was calculated to achieve a purification factor equivalent to that from cation exchange but with a lower concentration factor. While the cation exchange was favored on this basis, the two approaches were close enough that further optimization and economic analysis would be needed to be definitive.

Keywords:

• Dog gastric lipase
• extraction
• Triton X‐100
• corn
• endosperm
• aqueous two‐phase
• PEG
• cation exchange
• transgenic

Acknowledgements

This work was sponsored by the USDA CREES Grant Nos. 2003‐34496‐14026 and 2003‐34515‐14027. The authors thank Dian Octaviani, Steve Fox and Nathalie Vignaux at the Center for Crops Utilization Research of Iowa State University for supplying r‐DGL corn endosperm fraction and Qixin Zhong for suggesting cation‐exchange conditions.