溶菌酶的晶体培养试验

溶菌酶晶体培养实验

实验目的

近年来，X 射线晶体结构分析技术在受体研究中得到了广泛应用。 其关键是

获得具有高衍射分辨率的晶体。本试验通过溶菌酶晶体培养实验：

1. 了解蛋白质分子结晶的原理

2. 了解影响蛋白质分子结晶的因素

3.

掌握蛋白质晶体的悬滴培养法

蛋白质分子结晶的原理

]'igurc I . I. A lypicil solubility cunc lor a pr(Uei]i, ns a lun^iion nt ihc sail coiKcnti^itiun or aiuilher paiainukT. For Ivsl resulK (he urvslals should be 芒rvWn aL a lower level ol supers al oral ion than is required for formation or nuclei.

蛋白质结晶的4个主要步骤： 1.

测定蛋白质溶液的纯度

protein

concentration

solubility curve

parameter, e.g. salt concent ration

Figure 1.8. An ekclron

micrograph of n crystal face of tlie oxyjcn-Eransport ii 华 pitMciii hcinoey^Eiin from

Panuliru^ imermptus. The

nx>lecular weight of this pnMdn is 450.000； magnification: 250t 000x. (Courtesy of E.FJ. van Brugge IL }

2.蛋白质溶液与盐，有机化合物等沉淀剂混匀；膜蛋白需要加入去垢剂

3.混合溶液达到过饱和状态，形成晶核

4.晶核一旦形成，晶体开始生长

三. 影响蛋白质分子结晶的因素

1.待结晶蛋白质的纯度，一般要求纯度高于 95%

2.待结晶蛋白质的浓度，5~10 mg/ml

3.沉淀剂，盐，PEG，有机溶剂

4.pH 值

5.温度，4° C, 16° C

6.结晶方法，悬滴法，坐滴法

7?震动

四. 试剂及器材

1 ?试剂：溶菌酶蛋白，氯化钠，醋酸钠，去离子水

2?器材：可调移液器，晶体培养板，硅化盖玻片，真空脂，晶体培养箱，显微镜

五. 试验步骤

1.配制50 mg/ml溶菌酶溶液，0.1 M醋酸钠溶液，pH4.5

2.配制池液：(1) 5% (w/v)氯化钠，0.1 M醋酸钠溶液，pH4.5

(2) 8% (w/v)氯化钠，0.1 M醋酸钠溶液，pH4.5

3.在晶体培养板溶剂池中分别加入 0.3 ml池液(1)和池液(2)

4.在干净的硅化盖玻片上，滴加 2 ml溶菌酶溶液和2 ml池液

5.将盖玻片倒扣在加池液的有溶剂池上，用真空脂密封

6.将晶体培养板放入16° C晶体培养箱

7.第二天，在显微镜下观察晶体生长情况

I j^iiic 1.3. TJic lian^ing diop metluxl ul protein ciys(j||i/Ldii^i. I Wing unv with depressions. i he 卩rolein ion in suspended ns 訂drop t rum 3 gkiss cover ^lip shovel I K precipiLmi solution in a waled depression, (he glass slip is siliconized to prevent spreading of the drop. Equilibrium is reached dillusion ol vapor ftvin the drop 10 the preeipiuuing sole lion 01 vice versa. All of the depressions in the iray can. ol course, be used.

Procedure for Crystallization of Lysozyme

Chemicals needed:

Lysozyme protein

Sodium Chloride

Sodium Acetate buffer

Distilled water

Procedure

1.Prepare 50 mg/ml sample of lysozyme in 0.1 M NaAc pH4.5

2.Prepare well solutions:

a. 5% (w/v) NaCl, 0.1 M sodium acetate, pH4.5

b. 8% (w/v) NaCl, 0.1 M sodium acetate, pH4.5

3.Place 0.3 ml of well solution a, well solution b in each well of a VDX tray

l well solution 4.On a clean silated cover slide, create 4 drops of 2 卩l ysozyme

solution, 2

5.Seal these cover slips with grease over a well

6.Place the VDX plate in 16 °C incubator.

7.Observe the crystals next day under microscope