The Application Of Electrophoresis-chromatography Combined Technology In Purification Of Low-abundance Proteins

Free-flow electrophoresis is a full-liquid phase electrophoresis separation technology with no support medium,which has both analytical and preparative functions.In a free-flow electrophoresis system,charged solutes are laterally biased towards different streams depending on their electrophoretic mobility or isoelectric point(p Is).Compared with many electrophoresis techniques,free-flow electrophoresis has many merits:(i)continuous solute separation;(ii)high recovery due to absorbance matrix-free;(iii)high preservation of bioactivity due to mild aqueous circumstance fitting to requirement of enzyme and cell purification;(iv)low cost due to cheap buffers used as separation media;and(v)better resolution achieved in space.Owing to these merits,free-flow electrophoresis has been used for separations of numerous solutes,such as protein,organalles,DNA,chiral drug,cell membranes,malaria parasites and viruses.Free-flow electrophoresis was rarely used for the separation of solute with low-abundance,which had more important significance for bio-purification.In this work,herein free-flow electrophoresis was used to purify low-abundance protein for the first time.In the design of the free-flow zone electrophoretic separation,lysozyme in egg white was chosen as the model protein due to its extremely low abundance(less than 1.5%).In the experiment,lysozyme in egg white was first separated by free-flow zone electrophoresis,lyophilized and concentrated,and purified by gel filtration chromatography of Sephadex G50.In all experiments,a special polyacrylamide-acrylic acid copolymer(P(AM-co-AA))gel was used to characterize the protein and optimize the conditions for free-flow zone electrophoresis: 350 V,10 m A current limit,14? temperature control,130 ?L/min sample flow rate,4.9 m L/min carrier buffer flow rate,16.3m L/min electrode buffer circulation speed and 20 m M Tris-acetate buffer solution(p H 5.5),only two separation steps are required.We successfully purified low-abundance lysozyme(concentrationof about 1.4%)from its complex matrix to a high purity of 80%.Meanwhile,mass spectrometry was used to identify a molecular weight of approximately 16.2 k Da.Through standardized agarose diffusion experiments,we confirmed that the purified lysozyme has high antibacterial activity,indicating that free-flow zone electrophoresis has great potential for purifying low-abundance solutes in complex samples,which can promote its clinical and application in the pharmaceutical industry.