柱下酶切用酶的制作方法
1L TB菌体用PBS重悬至35ml做高压裂解，最终40ml总量。上清用0.5ml流速，挂3-4ml柱子，PBS漂洗30ml。用含有10%甘油的洗脱液洗脱，每管收集体积为2ml，只取主峰，可以粗略定量，也可以取百分之一的量在Akta上用上样泵来做积分定量（参考下面的积分定量方法举例）。也可以用分光光度计定量，一般可用稀释20倍定量。马上分装成0.25mg每管，可切5ml介质。 柱上酶切用酶的制作方法
亲和纯化方法同柱下酶切，洗脱液不含甘油，得到10ml左右洗脱液，浓缩10倍，再加入10ml 含有10%甘油和2mM DTT的PBS稀释，一共浓缩三次换buffer，每次浓缩时间一般在30分钟以内。最终浓缩至10mg/ml，浓缩10分钟混匀一次。尽快分装成0.25mg每管，可切5ml介质。 柱上酶切用酶的Q纯化制作方法
亲和纯化方法同柱下酶切，洗脱液不含甘油，收集后加水大于三分之一。过Q柱，AB液配方中含有10%甘油，8%B洗脱，50%B？直接洗下后积分定量，分装。 粗略定量参考
2000峰宽为10.5ml，蛋白质总量为24mg，取了12ml，浓度为2mg/ml，直接分装的话，可以用125ul和250ul每份。 积分定量方法举例 取10ul ppase浓缩液，用本底缓冲液（PBS）稀释到500ul，用上样泵做积分定量，积分体积为325mAu*ml，可知浓缩好的蛋白质光吸收为每毫升为32.5Au，光吸收为1，浓缩好的蛋白质浓度为32.5mg/ml，一共24mg蛋白。
表达
1. 取-80度保存的质粒pGEX-PPase转化BL21（DE3）感受态，涂布，37度孵箱培养12~16 h。 2. 挑取单克隆到50ml LBA培养基37度培养12-15小时。
3. 1%接种到1L TBA中，培养3小时，4度水浴降温，加终浓度为0.2mM的IPTG，15.3度诱导培养
18小时。
4. 4000rpm离心20分钟收菌。加入PBSE（1mM EDTA）至终体积30-40ml重悬。
纯化
1． 高压裂解2-3次。
2． 1.5万转离心30分钟，留上清。
3． PBSS平衡5ml的GST柱，上样，PBSS漂洗，4℃放置过夜，等RNA降解，再用含有10%甘油和
0.1% Tween20的酶切buffer漂洗干净。洗脱用含10%甘油的还原性谷胱甘肽溶液，2ml每管收集，取峰尖3*1.5ml。
4． 上样泵灌盐酸胍再生柱子，重力流灌水，灌20%乙醇，4℃保存柱子。
酶切效率确定
5ul，10ul，20ul酶切PH35C蛋白3-4天，电泳检测酶切产物，全部切开了。估计10ul过夜酶切1L的培养产物足够，下次实验再做验证。
保存
用PCR管，分装50ul每管，冻存于-80度。
使用
谷胱甘肽洗脱的融合蛋白，加入1份PPase，混匀后4度过夜酶切。
缓冲液
1． 10*PBSS（欧蒙基础）
配方：5包PBS粉剂（欧蒙），加入360ml 5M NaCl，定容到500ml，4℃保存。 2． 500ml 2M Tris（pH8.0），500ml 2M Tris（pH7.4）
350ml水溶解121.1g Tris碱，加转子搅拌或振荡下溶解（需要10分钟左右），加浓盐酸调pH至所需值，抽滤后4℃保存。
如温度升高，可加入预冷的水降温，温度下降1度pH升高0.03。pH6.8约需盐酸160 ml，pH7.4约需盐酸70 ml，7.6约需60ml，8.0约需42ml，8.0约需40ml。
Tris缓冲液稀释10倍pH下降0.1，故如稀释为20mM使用，则pH下降0.2。 3． 1L 2M Tris不调pH
800ml水溶解242.2g Tris碱，加转子搅拌，定容至1L，抽滤后4℃保存。 4． 500ml 0.5 mM EDTA
将93.05g二水乙二胺四乙酸二钠加入400ml水中，加入氢氧化钠颗粒调节pH（约需10g氢氧化钠颗粒），先加入7g左右，用搅拌棒彻底搅匀，在转子搅拌下，使其大部分溶解，静置一会儿，使气泡排出，并观察沉淀量。再继续搅拌，少量加入氢氧化钠颗粒，观察pH变化，勿使其变化过快，完全溶解后，加入4℃预冷的水（此时pH升高），继续调节pH为8.0，最终定容至1L，抽滤除去杂质，避光保存于4℃。 5． 10% Tween 20
2ml Tween 20，溶于20ml水，避光保存，千分之一稀释使用（根据GE资料）。 6． 250ml 20*酶切buffer储存液
400mM Tris-HCl (pH 7.0-pH 8.5), 3 M NaCl, 20 mM EDTA 配方：
2M Tris-HCL 50 ml 5M NaCL 150 ml 0.5M EDTA 10 ml
工作液成分（2M Tris稀释100倍使用，pH值会下降0.2）： 20mM Tris-HCl，150mM NaCl, 1 mM EDTA
作为漂洗液，使用前添加0.1% Triton X100（或者Tween20），做为酶切液使用前添加0.01% Triton（或者Tween20）和1mM DTT。 7． 100ml 20×还原性谷胱甘肽储液
成分：200mM还原型谷胱甘肽，50mM NaCl，1mM EDTA，0.01% Triton X-100（用欧蒙Tween20替代），100mM Tris（pH8.0） 50ml离心管1支，称取6.14g还原型谷胱甘肽，加20ml 5M NaCl，4ml 0.5M EDTA，0.2ml 10% Tween20再加水溶解至50ml，倒入100ml烧杯，再量取50ml 2M Tris（pH8.0），倒入100ml烧杯，搅拌溶解，滤器过滤至50ml离心管，1ml分装冻存于-20度。 8． 500ml 7M盐酸胍
称取334.4g盐酸胍，加水到450ml。定容，抽滤，室温保存。
PPase信息
1． PPase是人类鼻病毒蛋白酶Human rhinovirus B从11号氨基酸（G P N T E F A L S L
L R ），N端接入为BamH，尾端为EcoR。所用载体为pGEX4t 2． pGEX4t-HR14载体表达蛋白质序列
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSGGGPNTEFALSLLRKNIMTITTSKGEFTGLGIHDRVCVIPTHAQPGDDVLVNGQKIRVKDKYKLVDPENINLELTVLTLDRNEKFRDIRGFISEDLEGVDATLVVHSNNFTNTILEVGPVTMAGLINLSSTPTNRMIRYDYATKTGQCGGVLCATGKIFGIHVGGNGRQGFSAQLKKQYFVEKQ 3． 蛋白质参数
Number of amino acids: 410 Molecular weight: 46403.5
Theoretical pI: 6.80
Ext. coefficient 49195
Abs 0.1% (=1 g/l) 1.060, assuming all pairs of Cys residues form cystines Ext. coefficient 48820
Abs 0.1% (=1 g/l) 1.052, assuming all Cys residues are reduced
1L LB，OD 0.8左右诱导，收菌。 缓冲液配制：
1M 6.8的Tris稀释50倍（稀释10倍下降0.1），上纯化仪，温度为9.7度，pH为6.8，25度测pH值应该是6.35
1M 7.4的Tris稀释50倍，上纯化仪，温度为9.7度，pH为7.5，25度测pH值应该是7.05，所以酶切buffer可以用这个缓冲液稀释而成。
二者混合后，pH为7.25，温度升高1度，下降0.03，所以如果25度测的话，应该下降0.45，pH值为6.8
裂解Buffer：
40 mL of 50 mM Tris-HCl, 1 mM EDTA, 5 mM DTT at pH 8.8.
纯化方法：
4度30分钟之内，缓缓滴入1ml 10% PEI，2万转离心，上清上GST柱，用谷胱甘肽洗脱，用PPase Buffer（添加10%甘油）透析，浓缩到10 mg/ml，然后分装，10 ul相当于0.1 mg的酶可以切2ml介质。如果浓缩到1 mg/ml，100 ul分装即可。
500ml酶切buffer配方
1.21g Tris-HCl, 15ml 5 M NaCl, 1ml 500 mM EDTA, 调节pH 7.0，500ul 1M DTT使用时添加
20*酶切buffer配方：
400mM Tris-HCl (pH 7.0), 3 M NaCl, 20 mM EDTA, 20 mM dithiothreitol(DTT可以使用时添加） 100ml 配方（调pH到7.0） 2M Tris-HCL 20 ml 5M NaCL 60 ml 0.5M EDTA 4 ml
1L 储存液配方（调pH到7.0） 2M Tris-HCL 200 ml 5M NaCL 600 ml 0.5M EDTA 40 ml
10*酶切buffer配方（用1M Tis pH7.4）： 100ml 配方（不需要调pH） 1M Tris-HCL 20 ml 5M NaCL 30 ml 0.5M EDTA 2 ml
500 储存液配方（不需要调pH） 1M Tris-HCL 100 ml 5M NaCL 150 ml 0.5M EDTA 10 ml
Source:
Comes from Human Rhinovirus – HRV3C Protease. This is a cysteine protease.
Our version of this protease is an N-terminal His-GST- dual-tagged version that runs ~47KDa on SDS-PAGE. Can be removed by either GST- or Ni-affinity resins (WARNING: Need to remove DTT prior to Ni-resin use).
Recognition site:
LEVLFQ/GP is the standard site in most pGex vectors. Alternative sites can be cleaved. LE-P4-LFQ/GP /=cleaved site P4=Val Ala or Thr.
Standard Buffer: 20mM TRIS pH=7.0 150mM NaCl
1mM DTT or 5-10mM BME (TCEPT not tested) 0.5mM EDTA (optional)
Buffer: pH ranges from 7.0 to 8.5 seem optimal
Temperature: 4C is optimal but this enzyme will work at room temperature (4-15C). Salt: 30-500mM NaCl have been tested with no changes in proteolysis. Reducing agent: Required. Need 1mM DTT or 5-10mM BME (TCEP has not been tested). Not to exceed 2mM DTT.
EDTA: (0 to 10mM) Often required to minimize metal poisoning of this enzyme (ie Ni+2 off Ni-column may form adducts in presence of DTT). First dialyze against cleavage buffer +0.5mM EDTA prior to addition of protease.
Detergents (Triton X-100, Tween-20, NP-40, Nonidet) 0 - 1% (v/v).
Cleavage in solution
1. Following elution of the GST fusion protein from Glutathione Sepharose, dialyze the eluate extensively against Cleavage Buffer in order to remove reduced glutathione from the sample.
-Note: Alternatively, sample may be adjusted to 1X Cleavage Buffer by addition of the appropriate volume of 10X Cleavage Buffer.
2. Add 1 µl (2 units) of PreScission Protease for each 100 µg of fusion protein in the eluate. If the amount of fusion protein in the eluate has not been determined, add 40 µl (80 units) of PreScission Protease for each ml of Glutathione Sepharose bed volume* from which the fusion protein was eluted. Incubate at 5°C for 4 hours.**
3. Once digestion is complete, apply the sample to washed and equilibrated Glutathione Sepharose to remove the GST portion of the fusion protein and the PreScission Protease from the protein of interest. Detailed instructions for the purification of GST fusion proteins are provided in the GST Purification Modules (from GE 27-4570-01, -02, -03) or the GST Gene Fusion Manual, available on-line (GE Healthsciences).
*Bed volume is equal to 0.5X the volume of a 50% Glutathione Sepharose slurry used or 0.75X the volume of the original Glutathione Sepharose slurry supplied in the Bulk GST Purification Module. RediPack columns contain a 2 ml bed volume. MicroSpin columns have a 50 µl bed volume.
**More rapid cleavage may be achieved by adding a greater amount of PreScission Protease.
Note: Digestion may be improved by adding TritonTM X-100, TweenTM 20, NonidetTM, or NP40 to a concentration of 0.01%. Concentrations of these detergents up to 1% do not inhibit PreScission Protease.
During cleavage reactions, it is recommended that samples be removed at various time points and analyzed by SDS-PAGE to estimate the yield, purity, and extent of digestion. The amount of PreScission Protease, temperature and length of incubation required for complete digestion of a given GST fusion partner may vary depending on the fusion partner. Optimal conditions for each fusion should be determined in pilot experiments. Substrate recognition and cleavage are likely to be dependent not only upon primary structural signals, but also upon the secondary and tertiary structures of the fusion protein as well.
Units:
1Unit will cleave >90% of 100ug of protein on standard buffer at 4C in 16 hours. There is 833-1000 Units per mg of purified protease.
Some define the “Unit” as 1U will cleave 100mg of target and there are ~ 1U/mg
of protease. So if this is stored at 10mg/ml one only needs to use 100ul to cleave 100mg of protein.
Additives:
Things that do NOT inhibit this enzyme: Upto 2mM DTT 2% glycerol
0.1% triton X-100
Antipain dihydrochloride 74 µM Aprotinin 0.3 µM Bestatin 130 µM E-64 28 µM Leupeptin 1 µM Pepstatin 1 µM
Phosphoramidon 0.6 mM PMSF 1 mM ZnCl2 10 mM
Things that strongly inhibit this protease (>50% reduction): ZnCl2 100 mM
PefablocTM7 SC 4 mM Chymostatin 100 µM
Other Cysteine modifying reagents.
Storage:
Store at –20C in small aliquots. Long term at –80C.
Suppliers: Kathy
GE Lifesciences Accelagen
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