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【求助】小弟在表达纯化GST蛋白的时候遇到困难
【求助】小弟在表达纯化GST蛋白的时候遇到困难
我现在在纯化GST重组蛋白(表达是费了一个月的时间,好不容易搞定了,汗),遇到一个问题,请大家指教!
我的重组蛋白是不可溶的,加上GST为66KD,我是先把它挂上 sepharose 4B 的柱子,然后用thrombin切,我就是想问加thrombin的量怎么确定,就是要摸条件也要有一定的根据来计算,请知道的朋友赐教,谢谢!
查看完整版本请点击这里：
【求助】小弟在表达纯化GST蛋白的时候遇到困难
【求助】小弟在表达纯化GST蛋白的时候遇到困难
最新回复
小野花 (2014-5-24 11:21:53)
utt0989 (2014-5-24 11:22:14)
箭头儿 (2014-5-24 11:22:33)
按照柱上酶切的规程，GE的酶切用凝血酶，说明书上对于用量有专门的要求。你可以根据你柱子的体积计算出柱上酶切所需要的用量。
如果你的柱子很大，而凝血酶又不多，可以减少酶量，延长酶切时间。
你的蛋白不可溶，建议你先优化好表达条件，大多数GST蛋白是可以在胞内以可溶形式表达的。一般来说，很少去纯化变性的GST融合蛋白。
utt0989 (2014-5-24 11:22:52)
还有就是GST蛋白可以用尿素来溶解上柱的吗?能挂的上吗?
箭头儿 (2014-5-24 11:23:12)
QUOTE:
显然是不行的，变性条件下，蛋白全都线性化了，怎么能亲和呢？GST蛋白纯化方法都是对于天然蛋白而言的，his－tag蛋白是可以在变性条件下纯化的
小野花 (2014-5-24 11:23:33)
ＧＳＴ融合蛋白利用尿素溶解后，通过稀释复性或者透析复性至尿素的浓度低于２Ｍ后，都可以直接上柱进行亲和纯化．
不过还是建议你尽量优化各项表达条件，使蛋白以可溶性的方式表达为宜．
utt0989 (2014-5-24 11:23:58)
QUOTE:
请问你那里thrombin的说明书吗?utt0989 (2014-5-24 11:24:15)
我是用下面的方法做的,请大家看以下有什么不妥之处,谢谢!
GST融合蛋白的表达及纯化
（一）GST融合蛋白的表达
1、 将已构建好的pGEX质粒转化入BL-21菌株，挑单克隆菌落接种于5ml 2×YT (Amp)液体培养基中，37℃ 300rpm培养过夜。
2、 过夜饱和菌1׃100接种于100 ml 2×YT(Amp)中，37℃ 300rpm 培养至 OD600=0.6-0.8，1：1000加入IPTG，继续培养3-4h。
3、 8000 rpm离心10 min，去上清，称得湿菌体重，每克菌体以10ml STE重悬，离心8000 rpm离心10 min，去上清（所得菌体可在此步骤-20℃保存）。 （以上操作均冰上操作）
（二）GST融合蛋白的纯化
1、 每克菌体以10ml STE重悬，加入0.1体积的溶菌酶,冰浴15-60 min，间歇吹打菌体，使细胞壁破裂，至其呈粘稠状。
2、 加入1M DTT至终浓度为5mM，加入Sarkosyl至终浓度为1.5%。每15ml菌体冰浴超声2 min（6号变幅杆，200w ，打1秒停1秒），至菌体不再粘稠。
3、 12000rpm离心15min ，分装上清，-20℃储存。
4、 取一支冻存细胞裂解液溶解后，加入Triton X-100至终浓度为2%,置冰浴30mins后， 4℃ 12000rpm离心15min，取上清，然后直接纯化。
5、 加入20µl 50% Slurry， 4℃ 摇动混匀30min-120min。
6、 3000rpm离心5min，去上清，重悬于100µl PBS中，重复洗涤四次，去上清。
7、 加入6ml 1*PBS+130μl (1unit/μl) Thrombin 酶切过夜，室温（20℃左右）摇床上轻轻摇动。
8、 3000rpm,5’,或装柱收集，上清-80℃保存。
9、 OD280测值，样品1：100稀释。
箭头儿 (2014-5-24 11:24:43)
Calbiochem的一般纯度稍低，但也可以用来酶切
NOVAGEN的69671和amersham都专门用于酶切，效果更好一些，具体如何使用，他们会提供给你一个使用说明书，仔细看看再做。
给你两个网址
cuturl('http://www4.amershambiosciences.com/aptrix/upp01077.nsf/Content/Products?OpenDocument&parentid=38825&moduleid=38869&zone=Proteomics')
cuturl('http://www.merckbiosciences.com/Products/BrowseProductsByCategory.asp?catid=292')
都可查到
popo520 (2014-5-24 11:25:04)
破碎后上清要电泳检测一下啊，如果都是包涵体，上清根本没有你的东西啊！包涵体过不到柱上的
utt0989 (2014-5-24 11:25:22)
有做过GST不溶蛋白复性的把自己的protocal贴出来好吗,谢谢啦,大家共享嘛!
avi317 (2014-5-24 11:25:43)
上柱之前先测一下蛋白含量估计一下融合蛋白的量,凝血酶的用量好象是10U/mg.
箭头儿 (2014-5-24 11:26:07)
AFFINITY CHROMATOGRAPHY PURIFICATION OF GST FUSION PROTEIN FROM INCLUSION BODIES
ref：current protocols in protein science
In some cases, fusion proteins are entirely or primarily located in a denatured, aggregated
form in inclusion bodies. Glutathione-S-transferase (GST) fusion proteins can often be
purified from inclusion bodies after solubilization in urea or another denaturant followed
by renaturation by dialysis (UNIT 6.3). Other methods of solubilization include addition of
detergents such as Sarkosyl (N-laurylsarosine; Grieco et al., 1992; Frangioni, 1992). After
denaturation and renaturation, it is important to ensure that the protein has regained its
native conformation and function (UNITS 6.4 & 6.5). Although denatured GST will not bind
to the glutathione column and hence will not be recovered by this method, the possibility
that the GST moiety has refolded when the fusion partner has not folded properly should
also be considered. As an alternative to purifying proteins from inclusion bodies, growing
the cells at a lower temperature will often shift the fusion protein into the supernatant
while still producing ≥10 mg of fusion protein per liter of culture when pGEX vectors are
used (see Strategic Planning and see Troubleshooting). All steps should be performed in
a cold room at 4°C unless otherwise noted.
Additional Materials
U buffer
-----------------------------------
U buffer:
5 M urea
50 mM Tris base
5 mM EDTA
5 mM 2-mercaptoethanol (2-ME)
1 μg/ml leupeptin
1 μg/ml pepstatin
0.15 mM phenylmethylsulfonyl fluoride (PMSF) in isopropanol
1 mM diisopropyl fluorophosphate (DFP)
Adjust pH to 8.0 with 6 M HCl
Bring to final volume with cold H2O
Prepare fresh daily
CAUTION: DFP is a dangerous neurotoxin. Handle the neat reagent with double gloves in
a chemical fume hood only. Carefully follow all precautions supplied by the manufacturer
for this chemical.
---------------------------------------------------------------------------
箭头儿 (2014-5-24 11:26:55)
PBS/glycerol buffer
--------------------------
PBS/glycerol buffer
1× PBS (APPENDIX 2E)
20% (v/v) glycerol
1% (v/v) Triton X-100
5 mM 2-mercaptoethanol (2-ME)
5 mM EDTA
0.1 μg/ml leupeptin
0.1 μg/ml pepstatin
0.15 mM phenylmethylsulfonyl fluoride (PMSF) in isopropanol
0.1 mM diisopropyl fluorophosphate (DFP)
Adjust pH to 7.4 with 1 M NaOH
Bring to final volume with cold H2O
Prepare fresh daily
CAUTION: DFP is a dangerous neurotoxin. Handle the neat reagent with double gloves in
a chemical fume hood only. Carefully follow all precautions supplied by the manufacturer
for this chemical.
---------------------------
Low-speed refrigerated centrifuge (e.g., Beckman J6-B and JS-4.2 rotor or
equivalent), 4°C
Additional reagents and equipment for dialysis
1. Preequilibrate the glutathione column, lyse the cells, and separate the lysate pellet,
which includes the inclusion bodies, and supernatant (see Basic Protocol 2, steps 1
to 9).
2. Centrifuge washed pellet (see Basic Protocol 2, step 9) 20 min at 48,000 × g, 4°C.
Decant the supernatant and resuspend the pellet in 12 ml freshly prepared U buffer
per 600 ml original culture. Incubate 2 hr on ice.
Pellets should be resuspended in 20 l U buffer per ml of culture.
3. Centrifuge 20 min at 48,000 × g, 4°C. Carefully transfer the supernatant to a clean
50-ml centrifuge tube.
The extracted fusion protein should now be in the supernatant.
4. Add Triton X-100 to the supernatant to give a final concentration of 1% (v/v).
5. Dialyze the sample 2 to 3 hr in PBS/glycerol buffer.
Dialysis buffer volume should be 20 times the sample volume.
6. Dialyze sample overnight in PBS/EDTA/PMSF.
Dialysis buffer volume should be >100 times the sample volume.
7. Remove the sample from the dialysis bag and centrifuge 20 min at 4000 × g, 4°C.
8. Column purify the fusion protein (see Basic Protocol 2, steps 11 to 15).
pengke1983 (2014-5-24 11:27:14)
我的Q是345400628,信箱市wangxing5421@163.com.
还有,哪位哥哥姐姐知道MTPBS是什么,还有,洗柱子配方是什么,各种书众说纷纭,什么是最好.
pengke1983 (2014-5-24 11:27:33)
GST柱洗柱用的是6M盐酸胍，想想就知道能不能用8M尿素了。还有就是我不明白既然明明要用GST柱，为何不用可溶性表达呢？不溶蛋白应该用Ni柱比较好
utt0989 (2014-5-24 11:27:51)
MTPBS就是一种缓冲液,配方如下:
150mmol/L NaCl，16mmol/L Na2HPO4，4mmol/L Na2HPO4，pH7.4
【求助】小弟在表达纯化GST蛋白的时候遇到困难