集胞藻类金属硫蛋白的纯化、性质和溶液构象的研究

Studies on the Purification, Characterization and Conformation in Solution of Metallothionein-like Substance from Synechocystis 6803

集胞藻Synechocysitissp.PCC6803光照培养，加入100μmol/LZnCl2诱导藻内类金属硫蛋白（MT-like）的表达。离心收集鲜藻，超声波破碎细胞，离心除沉淀。上清液72℃加热3min去杂蛋白，离心，上清液依次经过分子筛层析柱，阴离子交换柱和脱盐柱，可得蓝藻类金属硫蛋白。5L培养液收集鲜藻7.6g，一次上样1．52g鲜藻的裂解物，冻干后得15mg纯化的蛋白样，为鲜藻重的0．1％．经分析其等电点为pH4．5，分子量为6．986kD，由58个氨基酸残基组成。其序列中含有较多疏水氨基酸残基（36％），但其半胱氨酸含量仅为5％。CD（圆二色性）图谱表明其二级结构主要为无规卷曲，不含α－helix和β-sheet，无哺乳动物的金属硫蛋白所具有的典型双结构域结构。紫外吸收光谱表明Zn结合的类金属硫蛋白在220nm也有较高的吸收值。红外光谱分析的结果表明，此类金属硫蛋白的吸收光谱与高等动物的金属硫蛋白的吸收光谱类似。

The cyanobacterium Sywechopstis sp. PCC 6803 was cultivated for 5 days in sterile liqUid mediumBG-11 at room temperature under fluorescent light, aerated by stirring under sterile condition. ZnCl2 was thenadded into the medium at final concentration of 50 μmol/L to induce the expression of the metallothionein-like(MT-like) compound. 5 days later, the ZnCl2 concentration was increased to 100 μmol/L. After 2 days, cellswere collected by centrifugation, then disrupted by ultrasonic wave. Aner centrifugation, the supernatant wastreated at 72℃ for 3 min to denature the unstable proteins. Again after centrifugation, the supernatant wasapplied to Sephacryl S-100 molecular sieve column, then DEAE-Sepharos F. F. ion exchange column andSephadex G-25 column for desalination. The Zn-binding flaction was frozen dried. In 5 L culture, 7.6 g wetcyanobacterial cells were obtained. Each time the lysate of 1. 52 g of them was processed, finally 1. 5 mg purified MT-like comPOund was obtained, which was 0. 1% of the total weight of wet cyanobacterial cells. Isoelectric focusing (IEF) showed that its pl was pH 4. 5. Mass spectrometry and amino acid compeition analysis indicated that the molecular weight of the protein was 6. 986 kD. Amino acid composition analysis showed that the content of hydrophobic amino acid residues was 36%, but the Cys content was not very high (only 5%).Ultraviolet absorption spectra showed that Zn-MT-like binding complex also had a high absothance at 220 nm.CD spectroscopy proved that the secondary structure of this protein was a random coil, and that there were no α-helix and β-sheet in the structure. FT-1R spectroscopy of cyanobacterial MT-like compound was similar to thatof the typical MT of mammal.