Expression, Purification And Biochemical Characteristic Analysis Of Human Coactosin-Like Protein

Using 5LO as the probe, CLP (coactosin-like protein), a new human protein, is obtained from human lung cDNA library, by yeast two-hybrid analysis. The mRNA of CLP is discovered in various tissues and is highly expressed in placenta, lung, kidney and peripheral blood lymphous leucocyte. Endogenous CLP mainly locates in cytoplasm of marrow cells. CLP shares high homology with actin-depolymerizing factor/cofilin (ADF) family which is widely distributed in eukaryotes. It contains LKKAET sequence fragment which is specific among other actin binding proteins, and shares extremely high homology with coactosin. Since 1996, Biochemical and structural analysis on CLP have been carried out. So far, it has been known that CLP up regulates the activity of 5LO, interacts with 5LO and actin via different binding sites. It participates in cytoskeleton movements, regulates the inflammatory response such as the chemotaxis, adhesion and phagocytosis of leucocytes, and induces tumor specific cellular and humoral immune response. But, there are still some unsolved puzzles about this protein. For example, little is known about the interaction mechanism between CLP, 5LO and F-action, how CLP regulates the movement of cells, and by what molecular mechanism CLP participates in the occurrence and development of some inflammatory and immune diseases such as tumor and asthma.The gene of CLP was PCR amplified from human liver cDNA library and cloned into the prokaryotic expression vector (pGEX-6p-1). The CLP protein was overexpressed and purified through affinity chromatography (Glutathione Sepharose? 4B) and gel filtration chromatography (Superdex 75). The acquired CLP protein with high purity was analyzed by SDS-PAGE, Dynamic Light Scattering and Analytical Ultracentrifuge were used to get further information. The results showed that the CLP protein was mainly monomer in solution. The friction factor was 1.909, which confirmed the potential linearization tendency of CLP and the extent of linearization was relatively high. The results suggested the potential linearization tendency of CLP and established a certain experimental basis for the constitution of an interaction model between CLP and F-actin.