蛋白质的分离与纯

1、纯化目的纯化目的: 研究其结构、组成、性质和功能等。研究其结构、组成、性质和功能等。 纯化方法的依据纯化方法的依据：蛋白质的基本性质：蛋白质的基本性质 1）溶解性：盐析等。）溶解性：盐析等。 2）分子大小和形状：凝胶过滤（分子筛过滤），）分子大小和形状：凝胶过滤（分子筛过滤），透析，超离心，超滤等。透析，超离心，超滤等。 3）电荷（酸碱性质）：电泳，离子交换层析，等）电荷（酸碱性质）：电泳，离子交换层析，等电聚焦等。电聚焦等。 4）吸附性质：吸附层析，疏水互作层析。）吸附性质：吸附层析，疏水互作层析。 5）特异结合亲和性：亲和层析。）特异结合亲和性：亲和层析。 有的方法利用了蛋白质几个方面的性

2、质。有的方法利用了蛋白质几个方面的性质。 一般要连续采用多种方法才能达到纯化的目一般要连续采用多种方法才能达到纯化的目的。的。Stabilization of proteins 蛋白质的稳定蛋白质的稳定 防止蛋白质变性或失活。防止蛋白质变性或失活。 措施：措施： 1）缓冲）缓冲pH； 2）低温；）低温；250C,一般一般40C; 3) 加蛋白酶抑制剂加蛋白酶抑制剂;Assay of proteins 蛋白质的检测蛋白质的检测 纯化过程中纯化过程中,不断检测目的蛋白质的存在和不断检测目的蛋白质的存在和纯度变化。纯度变化。 酶：检测其催化产生的产物。酶：检测其催化产生的产物。 非酶蛋白质：检测其生

3、物学效应，如抗原抗非酶蛋白质：检测其生物学效应，如抗原抗体反应。体反应。（一）盐析：（一）盐析：在蛋白质溶液中加入大量中性盐，以破坏蛋白质的在蛋白质溶液中加入大量中性盐，以破坏蛋白质的胶体性质，使蛋白质从溶液中沉淀析出，称为胶体性质，使蛋白质从溶液中沉淀析出，称为盐析盐析。依据：大多数蛋白质在高盐浓度时溶解度降低。依据：大多数蛋白质在高盐浓度时溶解度降低。 盐析对样品有浓缩作用。盐析对样品有浓缩作用。溶液中加入一定量的中性盐如溶液中加入一定量的中性盐如NHNH4 4 2 2SOSO4 4A A 由于由于 NHNH4 4 2 2SOSO4 4的亲水性比蛋白质亲水性大，能与大的亲水性比蛋白质亲水性

4、大，能与大量的水分子结合，使量的水分子结合，使蛋白质表面蛋白质表面水膜退化、消失；水膜退化、消失；B NH42SO4解离，中和了蛋白质颗粒所带的电荷，破坏解离，中和了蛋白质颗粒所带的电荷，破坏蛋白质表面电荷层而沉淀析出蛋白质表面电荷层而沉淀析出 Size-exclusion chromatography,also called gel filtration, separates proteins according to size. Thecolumn matrix is a cross-linked polymer with pores of selected size.Larger pro

5、teins migrate faster than smaller ones, because they are toolarge to enter the pores in the beads and hence take a more directroute through the column. The smaller proteins enter the pores andare slowed by their more labyrinthine path through the column.Ion-exchange chromatography exploits differenc

6、es in the sign and magnitude of the net electric charges of proteins at a given pH. The column matrix is a synthetic polymer containing bound charged groups; those with bound anionic groups are called cation exchangers, and those with bound cationic groups are called anion exchangers. Ion-exchange c

7、hromatography on a cation exchanger is shown here. The affinity of each protein for the charged groups on the column is affected by the pH (which determines the ionization state of the molecule) and the concentration of competing free salt ions in the surrounding solution. Separation can be optimize

8、d by gradually changing the pH and/or salt concentration of the mobile phase so as to create a pH or salt gradient.(c) Affinity chromatography separates proteins by their binding specificities. The proteins retained on the column are those that bind specifically to a ligand cross-linked to the beads

9、. (In biochemistry, the term “ligand” is used to refer to a group or molecule that binds to a macromolecule such as a protein.) After proteins that do not bind to the ligand are washed through the column, the bound protein of particular interest is eluted (washed out of the column) by a solution con

10、taining free ligand.特点特点:1)1)使用的固定相支持剂颗粒很细使用的固定相支持剂颗粒很细, ,表面积很大表面积很大.2).2)溶剂系统采取高压溶剂系统采取高压, ,洗脱速度增大洗脱速度增大.3).3)高分辨率高分辨率反相反相HPLC(reversed-phase HPLC):HPLC(reversed-phase HPLC):固定相是非极固定相是非极性的烷基硅烷与硅胶形成的键合相，与被分离物只性的烷基硅烷与硅胶形成的键合相，与被分离物只可能有疏水作用；流动相是相对极性的可能有疏水作用；流动相是相对极性的High performance liquid chromatogra

11、phy 高效液相层析高效液相层析(HPLC)(1)A modern refinement in chromatographic methods is HPLC, or high-performance liquid chromatography. HPLC makes use of high-pressure pumps that speed the movement of the protein molecules down the column, as well as higher-quality chromatographic materials that can withstand t

12、he crushing force of the pressurized flow. By reducing the transit time on the column, HPLC can limit diffusional spreading of protein bands and thus greatly improve resolution. Electrophoresis.Different samples are loaded in wells or depressions at the top of the polyacrylamide gel. The proteins move into the gel when an electric field is applied. The gel minimizes convection current