title: Single-step purification of recombinant proteins using elastin-like peptide-mediated inverse transition cycling and self-processing module from Neisseria meningitides FrpC.
abstract: Purification of recombinant proteins is a major task and challenge in biotechnology and medicine. In this paper we report a novel single-step recombinant protein purification system which was based on elastin-like peptide (ELP)-mediated reversible phase transition and FrpC self-processing module (SPM)-mediated cleavage. After construction of a SPM-ELP fusion expression vector, we cloned the coding sequence for green fluorescent protein (GFP), the Fc portion of porcine IgG (pFc) or human β defensin 3 (HBD3) into the vector, transformed the construct into Escherichia coli, and induced the fusion protein expression with IPTG. The target-SPM-ELP fusion proteins GFP-SPM-ELP, Fc-SPM-ELP and HBD3-SPM-ELP were expressed in a soluble form and efficiently purified from the clarified cell extracts by two rounds of inverse transition cycling (ITC). Under the optimized conditions, the SPM-mediated cleavage efficiencies for the three fusion proteins ranged from 92% to 93%. After an additional round of ITC, the target proteins GFP, pFc and HBD3 were recovered with purities ranging from 90% to 100% and yields ranging from 1.1 to 36mg/L in shake flasks. The endotoxin levels in all of the three target proteins were <0.03EU/mg. The three target proteins were functionally active with the expected molecular weights. These experimental results confirmed the high specificity and efficiency of SPM-mediated cleavage, and suggested the applicability of SPM-ELP fusion system for purification of recombinant proteins.
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China.
authors.forename: Wen-Jun
authors.initials: WJ
authors.lastname: Liu
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wu
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China.
authors.forename: Bi
authors.initials: B
authors.lastname: Xu
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China.
authors.forename: Xin-Yu
authors.initials: XY
authors.lastname: Zhang
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China.
authors.forename: Xiao-Li
authors.initials: XL
authors.lastname: Xia
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China. Electronic address: sunh@yzu.edu.cn.
authors.forename: Huai-Chang
authors.initials: HC
authors.lastname: Sun
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D004549:Elastin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C083530:frpC protein, Neisseria meningitidis; D004549:Elastin
keywords: Elastin-like peptide; Fusion system; Protein purification; Self-processing module