Zheng Hai, Miyakawa Takuya, Sawano Yoriko, Yamagoe Satoshi, Tanokura Masaru
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.
Protein Expr Purif. 2013 Apr;88(2):221-9. doi: 10.1016/j.pep.2013.01.008. Epub 2013 Jan 19.
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.
人白细胞衍生趋化因子2（LECT2）是一种针对中性粒细胞的趋化因子，是一种16 kDa的分泌蛋白，由133个氨基酸组成，含有三个分子内二硫键。在此，我们提出了一种使用高静水压（HHP，200 MPa）重折叠技术制备人LECT2的有效方法。当LECT2在大肠杆菌细胞中过表达时，大多数LECT2分子形成不溶性包涵体（IBs）。将HHP应用于从不溶性IBs中重折叠LECT2，这显著提高了活性LECT2的产量。圆二色光谱（CD）和核磁共振（NMR）测量表明，重折叠的LECT2具有与可溶性表达的LECT2难以区分的三级结构。此外，重折叠的LECT2和可溶性表达的LECT2表现出相同水平的趋化活性。
