Automated flow-based anion-exchange method for high-throughput isolation and real-time monitoring of RuBisCO in plant extracts

Abstract

[eng] In this work, a miniaturized, completely enclosed multisyringe-flow system is proposed for high-throughput purification of RuBisCO from Triticum aestivum extracts. The automated method capitalizes on the uptake of the target protein at 4ºC onto Q-Sepharose Fast Flow strong anion-exchanger packed in a cylindrical microcolumn (105 × 0.4 mm) followed by a stepwise ionic-strength gradient elution (0-0.8 mol/L NaCl) to eliminate concomitant extract components and retrieve highly purified RuBisCO. The manifold is furnished downstream with a flow-through diode-array UV/Vis spectrophotometer for real-time monitoring of the column effluent at the protein-specific wavelength of 280 nm to detect the elution of RuBisCO. Quantitation of RuBisCO and total soluble proteins in the eluate fractions were undertaken using polyacrylamide gel electrophoresis (PAGE) and the spectrophotometric Bradford assay, respectively. A comprehensive investigation of the effect of distinct concentration gradients on the isolation of RuBisCO and experimental conditions (namely, type of resin, column dimensions and mobile-phase flow rate) upon column capacity and analyte breakthrough was effected. The assembled set-up was aimed to critically ascertain the efficiency of preliminary batchwise pre-treatments of crude plant extracts (viz., polyethylenglycol (PEG) precipitation, ammonium sulphate precipitation and sucrose gradient centrifugation) in terms of RuBisCO purification and absolute recovery prior to automated anion-exchange column separation. Under the optimum physical and chemical conditions, the flow-through column system is able to admit crude plant extracts and gives rise to RuBisCO purification yields better than 75%, which might be increased up to 96 ± 9% with a prior PEG fractionation followed by sucrose gradient step.