Soluble expression, purification, and characterization of recombinant human flotillin-2 (reggie-1) in Escherichia coli.
Large scale production of recombinant human flotillin-2 (reggie-1) is desirable for structural and biochemical studies. However, as the major lipid rafts specific hydrophobic protein, flotillin-2 was difficult to be expressed as soluble and functional form in prokaryotic system. In this study, we first cloned and expressed human flotillin-2 in Escherichia coli with five different fusion tags: poly-histidine, glutathione S-transferase (GST), thioredoxin (TRX), N-Utilization substance (NusA) and maltose binding protein (MBP). We screened the expression level and solubility of the five flotillin-2 fusion proteins, the best MBP tagged flotillin-2 was then large scale produced. The optimized purification procedure included two steps of chromatography: Ni-NTA affinity chromatography and anion exchange chromatography. The typical yield was 36.0 mg soluble and functional recombinant flotillin-2 from 1 L of culture medium with purity above 97%. The activity of recombinant flotillin-2 was verified by pull-down assay with flotillin-1, showing that the purified recombinant flotillin-2 can specifically interact with flotillin-1. The circular dichroism (CD) spectroscopy showed that recombinant flotillin-2 had a very stable secondary structure dominated by α-helix, β-turn and random structure.
Song
Jiaping
J
Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200433, China.
Chen
Wentao
W
Lu
Zhisheng
Z
Hu
Xiaojian
X
Ding
Yu
Y
D015153:Blotting, Western; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility
C471213:His-His-His-His-His-His; D008565:Membrane Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C106354:flotillins; D006639:Histidine