Evaluating extraction and storage of a recombinant protein produced in agroinfiltrated lettuce.
Extraction and storage of a recombinant protein produced by transient expression following agroinfiltration of lettuce were investigated. Lettuce leaves expressing beta-glucuronidase (GUS) were extracted by homogenization in several buffer combinations, and the yield and stability were assessed. The reducing agent dithiothreitol (DTT) was found to be the most important (significant) component in the extraction buffer. An extraction buffer consisting of 50 mM sodium phosphate at pH 7.0 with 10 mM DTT produced a good yield and stabilized GUS. Leaching of GUS through intact agroinfiltrated lettuce leaves was determined to be infeasible, with a maximum flux of 10 microg GUS/h/m2 and recovery of 1.7% of the GUS content in 24 h. Freeze-drying was evaluated as a method to extend the shelf life of the perishable leaf material containing GUS. First- and second-order kinetic models and the Weibull distribution were compared to describe inactivation of GUS in the freeze-dried leaves and leaf extracts. The first-order model best fit the inactivation data. An Arrhenius model fit the first-order inactivation data with respect to temperature with R2 = 1.00. Freeze-drying the lettuce leaves extended the estimated half-life of GUS to 69 days at 21 degrees C versus 11 days at 4 degrees C for fresh lettuce.
Joh
Lawrence D
LD
Department of Biological and Agricultural Engineering, University of California, Davis, One Shields Avenue, Davis, California 95616, USA.
McDonald
Karen A
KA
VanderGheynst
Jean S
JS
D004229:Dithiothreitol / Q000494:pharmacology; D005612:Freeze Drying; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005966:Glucuronidase / Q000096:biosynthesis* / Q000187:drug effects / Q000302:isolation & purification*; D066298:In Vitro Techniques; D018545:Lactuca / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D008956:Models, Chemical; D010710:Phosphates / Q000494:pharmacology; D018515:Plant Leaves / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000302:isolation & purification*
D010710:Phosphates; D011994:Recombinant Proteins; D005966:Glucuronidase; C018279:sodium phosphate; D004229:Dithiothreitol