title: Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.
abstract: Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.
authors.affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, 541642, P. R. China.|Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou, 541642, P. R. China.
authors.forename: Hua-zhen
authors.initials: HZ
authors.lastname: Wang
authors.affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, 541642, P. R. China.|Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou, 541642, P. R. China.
authors.forename: Zhi-zhan
authors.initials: ZZ
authors.lastname: Chu
authors.affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, 541642, P. R. China.|Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou, 541642, P. R. China.
authors.forename: Chang-chao
authors.initials: CC
authors.lastname: Chen
authors.affiliation: Department of Pesticides, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Ministry of Agriculture, Beijing, 100193, China.|State Key Laboratory for Biology of Plant Diseases and Insect Pests, Beijing, 100193, China.
authors.forename: Ao-cheng
authors.initials: AC
authors.lastname: Cao
authors.affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, 541642, P. R. China.|Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou, 541642, P. R. China.
authors.forename: Xin
authors.initials: X
authors.lastname: Tong
authors.affiliation: Department of Pesticides, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Ministry of Agriculture, Beijing, 100193, China.|State Key Laboratory for Biology of Plant Diseases and Insect Pests, Beijing, 100193, China.
authors.forename: Can-bin
authors.initials: CB
authors.lastname: Ouyang
authors.affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, 541642, P. R. China.|Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou, 541642, P. R. China.
authors.forename: Qi-hang
authors.initials: QH
authors.lastname: Yuan
authors.affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, 541642, P. R. China.|Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou, 541642, P. R. China.
authors.forename: Mi-nan
authors.initials: MN
authors.lastname: Wang
authors.affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, 541642, P. R. China.|Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou, 541642, P. R. China.
authors.forename: Zhong-kun
authors.initials: ZK
authors.lastname: Wu
authors.affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, 541642, P. R. China.|Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou, 541642, P. R. China.
authors.forename: Hai-hong
authors.initials: HH
authors.lastname: Wang
authors.affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, 541642, P. R. China.|Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou, 541642, P. R. China.
authors.forename: Sheng-bin
authors.initials: SB
authors.lastname: Wang
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism; D005941:Glucokinase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D000516:alpha-Amylases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005941:Glucokinase; D000516:alpha-Amylases; D010450:Endopeptidases; C099488:TEV protease