title: Sequential tentacle grafting and charge modification for enhancing charge density of mono-sized beads for facilitated protein refolding and purification from inclusion bodies.
abstract: We have previously found that addition of like-charged media in a refolding solution can greatly enhance the refolding of pure proteins by suppressing protein aggregation. Herein, negatively charged mono-sized microspheres with sulfonic groups were fabricated to explore the facilitating effect of like-charged media on the refolding of enhanced green fluorescent protein (EGFP) expressed as inclusion bodies (IBs). A sequential polymer-tentacle grafting and sulfonate modification strategy was developed to increase the charge density of mono-sized poly(glycidyl methacrylate) (pGMA) beads (2.4μm). Namely, GMA was first grafted onto the beads by grafting polymerization to form poly(GMA) tentacles on the pGMA beads, and then the epoxy groups on the tentacles were converted into sulfonic groups by modification with sodium sulfite. By this fabrication strategy, the charge density of the beads reached 793μmol/g, about 2.8 times higher than that modified without prior grafting of the pGMA beads (285μmol/g). The negatively charged beads of different charge densities were used for facilitating the refolding of like-charged EGFP from IBs. The refolding yield as well as refolding rate increased with increasing charge density. The anti-aggregation effects of urea and like-charged microspheres were synergetic. In addition, partial purification of EGFP was achieved because the ion-exchange adsorption led to 52% removal of positively charged contaminant proteins in the refolded solution. Finally, reusability of the tentacle beads was demonstrated by repetitive EGFP refolding and recovery cycles.
authors.affiliation: Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China.
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Dong
authors.affiliation: Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
authors.forename: Ran
authors.initials: R
authors.lastname: Chen
authors.affiliation: Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China; Department of Bioengineering, School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, China. Electronic address: ycy@tju.edu.cn.
authors.forename: Chun-Yan
authors.initials: CY
authors.lastname: Yang
authors.affiliation: Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China. Electronic address: ysun@tju.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Sun
mesh_terms: D000327:Adsorption; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry* / Q000378:metabolism; D007474:Ion Exchange; D008863:Microspheres; D011109:Polymethacrylic Acids; D058849:Protein Refolding
chemical_list: D011109:Polymethacrylic Acids; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C042535:polyglycidyl methacrylate
keywords: Modification; Mono-sized microsphere; Negatively charged surface; Protein refolding; Purification; Tentacle bead