کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2020544 | 1069187 | 2013 | 9 صفحه PDF | دانلود رایگان |
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.
► Most LECT2 is expressed as inclusion bodies with an E. coli over-expression system.
► High hydrostatic pressure (200 MPa) refolds LECT2 from inclusion bodies.
► The refolded and solubly expressed LECT2 have the same structure and activity.
► The flexibility of the LECT2 structure is more restricted upon zinc ion binding.
► The chemotactic activity of LECT2 is insensitive to zinc ion.
Journal: Protein Expression and Purification - Volume 88, Issue 2, April 2013, Pages 221–229