title: Expression, high-pressure refolding and purification of human leukocyte cell-derived chemotaxin 2 (LECT2).
abstract: Human leukocyte cell-derived chemotaxin 2 (LECT2) is a chemotactic factor for neutrophils and a 16-kDa secreted protein consisting of 133 amino acids with three intramolecular disulfide bonds. Here, we propose an efficient method for the preparation of human LECT2 using a high hydrostatic pressure (HHP, 200 MPa) refolding technique. When LECT2 was over-expressed in Escherichia coli cells, most of LECT2 molecules became insoluble inclusion bodies (IBs). HHP was applied to the refolding of LECT2 from insoluble IBs, which dramatically improved the yield of the active LECT2. CD and NMR measurements demonstrated that the refolded LECT2 had a tertiary structure indistinguishable from the solubly expressed LECT2. In addition, both the refolded and solubly expressed LECT2 showed the same level of chemotactic activity.
authors: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors: Hai
authors: H
authors: Zheng
authors: Takuya
authors: T
authors: Miyakawa
authors: Yoriko
authors: Y
authors: Sawano
authors: Satoshi
authors: S
authors: Yamagoe
authors: Masaru
authors: M
authors: Tanokura
mesh_terms: D002633:Chemotaxis; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D006874:Hydrostatic Pressure; D002479:Inclusion Bodies / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D015032:Zinc / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D036341:Intercellular Signaling Peptides and Proteins; C103468:LECT2 protein, human; D011994:Recombinant Proteins; D015032:Zinc