Institution: | 1. Department of Instrument Science and Engineering, School of Electronic Information & Electrical Engineering, Shanghai Jiao Tong University, Shanghai, P. R. China
State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, P. R. China
The first three authors have contributed equally to the work.;2. College of Biological Sciences, China Agricultural University, Beijing, P. R. China
State Key Laboratory of Agro-biotechnology, China Agricultural University, Beijing, P. R. China
The first three authors have contributed equally to the work.;3. Department of Instrument Science and Engineering, School of Electronic Information & Electrical Engineering, Shanghai Jiao Tong University, Shanghai, P. R. China;4. State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, P. R. China;5. School of Physics and Astronomy, Shanghai Jiao Tong University, Shanghai, P. R. China;6. College of Biological Sciences, China Agricultural University, Beijing, P. R. China |
Abstract: | As an effective separation tool, free-flow electrophoresis has not been used for purification of low-abundance protein in complex sample matrix. Herein, lysozyme in complex egg white matrix was chosen as the model protein for demonstrating the purification of low-content peptide via an FFE coupled with gel fitration chromatography (GFC). The crude lysozyme in egg while was first separated via free-flow zone electrophoresis (FFZE). After that, the fractions with lysozyme activity were condensed via lyophilization. Thereafter, the condensed fractions were further purified via a GFC of Sephadex G50. In all of the experiments, a special poly(acrylamide- co-acrylic acid) (P(AM-co-AA)) gel electrophoresis and a mass spectrometry were used for identification of lysozyme. The conditions of FFZE were optimized as follows: 130 μL/min sample flow rate, 4.9 mL/min background buffer of 20 mM pH 5.5 Tris-Acetic acid, 350 V, and 14 °C as well as 2 mg/mL protein content of crude sample. It was found that the purified lysozyme had the purity of 80% and high activity as compared with its crude sample with only 1.4% content and undetectable activity. The recoveries in the first and second separative steps were 65% and 82%, respectively, and the total recovery was about 53.3%. The reasons of low recovery might be induced by diffusion of lysozyme out off P(AM-co-AA) gel and co-removing of high-abundance egg ovalbumin. All these results indicated FFE could be used as alternative tool for purification of target solute with low abundance. |