Secreted Expression Of Angiopoietin-2 In Pichia Pastoris And Its Funtion

Angiogenesis that plays an important role in embryogenesis, wound healing, tumorigenesis, is the process of generating new capillary blood vessels. It is strictly controlled by a balance between positive and negative factors .Among the numerous factors, the family of angiopoietins plays specific roles in angiogenesis. Angiopoietin-2, one member of the family,which participates in both normal and pathological Angiogenesis,is involved in many diseases about angiogenesis such as tumors,artherosclerosis,asthma.Angiopoietin-2 has characteristic protein structure of angiopoietins that contains coiled-coil domains in the amino-terminal region and a fibrinogen-like domain in the carboxy-terminal region. The fibrinogen-like domain may primarily have the function of receptor binding. Angiopoietin-2 has been originally described as an antagonist of Tie2 through inhibition of angiopoietin-2-induced phosphorylation of Tie2. However, recent reports indicate that angiopoietin-2 acts as agonist or antagonist to Tie2 in a context-dependent manner. The ability of angiopoietin-2 to stimulate the phosphorylation of Tie2 receptor depends on its concentration. Angiopoietin-2 at high concentration leads to the phosphorylation of Tie2 receptor,while angiopoietin-2 at low concentration does not.With an increasing interest in angiopoietin-2, there are reports of angiopoietin-2 expression in many systems. But angiopoietin-2 was mostly expressed either in insect cells using a baculovirus, or in mammalian cells such as COS cells, rat aortic A10 smooth muscle cells(A10 SMC), and human embryonic kidney (HEK) 293T cells. Most expressions were transient and low-level. Its coiled-coil domain, which is prone to aggregation and insolubility, may be the main reason.Here, we accomplished a high-level, stable expression of recombinant rat Ang2 with a carboxy-terminal His-tag in Pichia.pastoris. The gene of rat angiopoietin-2 was amplified, and then was inserted into the vector pBV220. The recombinant vector was transformed into E.coli. The gene couldn′t be expressed in E.coli. The gene of rat angiopoietin-2 then was inserted into the vector pPICZɑA, and the recombinant vector was transformed into Pichia.pastoris KM71H and GS115.After the transformation and screening ,we got the high expression strains K1.By optimizing the cultural condition for recombinant strain of K1, the optimal ferment conditons were established. Angiopoietin-2 was purified by His affinity chromatography. The cell assays showed that the recombinant protein at high level(2μg /ml)could increase the growth of of ECV304,while it at low leve(l0~0.2μg /ml)could not. Our research might provide a basis for further study on the function of angiopoietin-2.To study the other functions of angiopoietin-2, we amplified the human angiopoietin-2.The gene was ligated with the yeast two-hybrid vector pAS2-1. After the transformation into the yeast Y190 strain, we probed the expression of angiopoietin-2 through Western blot. Finally, angiopoietin-2 was used as the bait to screen a human fetal liver cDNA library for its interacters. Our research might provide an opportunity to find interacters of angiopoietin-2.