Controlled oxidative protein refolding using an ion-exchange column

Column-based refolding of complex and highly disulfide-bonded proteins simplifies protein renaturation at both preparative and process scale by integrating and automating a number of operations commonly used in dilution refolding. Bovine serum albumin (BSA) was used as a model protein for refolding and oxido-shuffling on an ion-exchange column to give a refolding yield of 55 % after 40 Ih incubation. Successful on-column refolding was conducted at protein concentrations of up to 10 mg/ml and refolded protein, purified from misfolded forms, was eluted directly from the column at a concentration of 3 mg/ml. This technique integrates the dithiothreitol removal, refolding, concentration and purification steps, achieving a high level of process simplification and automation, and a significant saving in reagent costs when scaled. Importantly, the current result suggests that it is possible to controllably refold disulfide-bonded proteins using common and inexpensive matrices, and that it is not always necessary to control protein-surface interactions using affinity tags and expensive chromatographic matrices. Moreover, it is possible to strictly control the oxidative refolding environment once denatured protein is bound to the ion-exchange column, thus allowing precisely controlled oxido-shuffling. (c) 2005 Elsevier B.V. All rights reserved.

Simulated rainwater effects on anion exchange capacity and nitrate retention in Ferrosols

Nitrate (NO3) accumulations (up to 1880 kg NO3-N/ha for a 12-m profile) in the soils of the Johnstone River catchment (JRC) may pose a serious environmental threat to the Great Barrier Reef lagoon if the NO3 were released. The: leaching of artificial rainwater through repacked soil columns was investigated to determine the effect of low NO3/low ionic strength inputs on the NO3 Chemistry of the JRC profiles. Repacked soil columns were used to simulate the 11.5-m profiles, and the soil solution anion and cation concentrations were monitored at 10 points throughout the soil column. As the rainwater was applied, NO3 leached down the profile, with substantial quantities exiting the columns. Anion exchange was discounted as the major mechanism of NO3 release due to the substantial net loss of anions from the system (up to 2740 kg NO3-N/ha over the experimental period). As the soils were dominated by variable charge minerals, the effect of changing pH and ionic strength on the surface charge density was investigated in relation to the release of NO3 from the exchange. It was concluded that the equilibration of the soil solution with the low ionic strength rainwater solution resulted in a lessening of both the positive and negative surface charge. Nitrate was released into the soil solution and subsequently leached due to the lessening of the positive surface charge. Loss of NO3 from the soil profile was slow, with equivalent field release times estimated to be tens of years. Although annual release rates were high in absolute terms (up to 175 kg NO3-N/ha.year), they are only slightly greater than the current loss rates from fertilised sugarcane production (up to 50 kg NO3-N/ha.year). In addition to this, the large-scale release of NO3 from the accumulations will only occur until a new equilibrium is established with the input rainwater solution.