Isolation and purification of natural tissue inhibitor of metalloproteinase-3 from human placenta
- VernacularTitle:人胎盘天然金属蛋白酶抑制因子3的分离纯化
- Author:
Jun XU
;
Peng WU
;
Lin HAN
;
Zhong ZHANG
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2006;10(29):182-183
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Matrix metalloproteinase (MMP) is a kind of protease family, its activity can be inhibited by tissue inhibitor of metalloproteinase (TIMP), especially by TIMP-3.OBJECTIVE: To fully isolate and purify natural TIMP-3, and to create enzyme-linked immunoassay of TIMP-3.DESIGN: Single-sample observation SETTING: Central Laboratory of Shenyang Medical College MATERIALS: Human placenta from Department of Obstetrics & Gynecology, Fengtian Hospital Affiliated to Shenyang Medical College (Informed consent was obtained from the relatives of patients) and MMP-1 from Research Institute. Fuji Chemical Industries, Ltd.METHODS: This experiment was conducted in the Central Laboratory of Shenyang Medical College between March 2001 and May 2002. Firstly,4 mol/L urea Tris-buffer solution (pH8.0) was used to prepare homogenate solution of placenta. Secondly, homogenate solution was performed chromatography through CM52 positive ion-exchange resin and Sephacryl S200 gel filtration. Thirdly, relative molecular weight and purity were detected by SDS- polyacrylamidedel gel electrophoresis (SDS-PAGE).Fourthly, Western blotting was used to identify the characters of purified protein. Fifthly, the inhibitory rate of TIMP-3 to MMP-1 was measured with immumofluorescence method.MAIN OUTCOME MEASURES: ① The relative molecular mass of protein showed by PAGE. ② Results of Western blot. ③ The inhibitory rate of TIMP-3 for MMP-1. ④ The recovery rate of TIMP-3 following purification.RESULTS: ①The isolated and purified TIMP-3 from human placenta consisted of non-glycosylated and glycosylated protein, with relative molecular mass of 24 000 and 27 000, respectively. ② The inhibitory concentration of non-glycosylated and glycosylated TIMP-3 was 1.1×1010 mol/L and 1.2×1010 mol/L respectively to MMP-1. The inhibitory concentration of placenta-derived TIMP-3 was significantly higher than that of recombinant TIMP-3 for MMP-1. ③ The recovery rate of TIMP-3 was 23.4% following two-step chromatography.CONCLUSION: Extracellular matrix of human placenta-derived TIMP-3 consists of non- glycosylated protein and glycosylated protein; Two kinds of purified TIMPs-3 have remarkable inhibitory concentration for MMP-1, and significantly higher in comparison with recombinant metalloproteinase inhibitory factor-3.
