Expression, purification, and characterization of recombinant human flotillin-1 in Escherichia coli.
Human flotillin-1 (reggie-2), a major hydrophobic protein of biomembrane microdomain lipid rafts, was cloned and expressed in Escherichia coli with four different fusion tags (hexahistidine, glutathione S-transferase, NusA, and thioredoxin) to increase the yield. The best expressed flotillin-1 with thioredoxin tag was solubilized from inclusion bodies, first purified by immobilized metal affinity column under denaturing condition and direct refolded on column by decreasing urea gradient method. The thioredoxin tag was cleaved by thrombin, and the flotillin-1 protein was further purified by anion exchanger and gel filtration column. The purified protein was verified by denaturing gel electrophoresis and Western blot. The typical yield was 3.4 mg with purity above 98% from 1L culture medium. Using pull-down assay, the interaction of both the recombinant flotillin-1 and the native flotillin-1 from human erythrocyte membranes with c-Cbl-associated protein or neuroglobin was confirmed, which demonstrated that the recombinant proteins were functional active. This is the first report describing expression, purification, and characterization of active recombinant raft specific protein in large quantity and highly purity, which would facilitate further research such as X-ray crystallography.
Ding
Yu
Y
Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai, China.
Jiang
Ming
M
Jiang
Weihua
W
Su
Yang
Y
Zhou
Hanqing
H
Hu
Xiaojian
X
Zhang
Zhihong
Z
D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015870:Gene Expression / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D005914:Globins / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006801:Humans; D007526:Isoelectric Point; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D000078787:Neuroglobin; D010445:Peptide Elongation Factors / Q000235:genetics; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D035602:Transcriptional Elongation Factors
D003598:Cytoskeletal Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D010445:Peptide Elongation Factors; D011994:Recombinant Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C106354:flotillins; C068727:nusA protein, E coli; C033223:polyhistidine; D006639:Histidine; D013879:Thioredoxins; D005914:Globins; D005982:Glutathione Transferase