(7.4)--His-tag重组蛋白纯化方法.pdf

His-tag重组蛋白纯化方法

【基本试剂】

1.Luria-Bertani(LB)mediumperlitre:10gtryptone(Oxoid),5gyeastextracts(Oxoid),5g

NaCl.AdjusttopH7.2withNaOH.Autoclaveandstoreat4°C.

2.IPTG,100mMstock:dissolve2.38gIPTGin100mLdeionisedwater.Filtersteriliseandstore

at−20°C.

3.Glucose:20%(w/v)D-glucosesolutionindeionisedHO.Autoclaveandstoresterilesolution

2

atroomtemperature.AddglucosetoLBmediumwithantibioticstoafinalconcentrationof

0.5-1%.

4.NativeSolutions：

A.Lysisbuffer(1L):50mMNaH2PO4;6.90gNaH2PO4·H2O(MW137.99g/mol),300mM

NaCl;17.54gNaCl(MW58.44g/mol),10mMimidazole;0.68gimidazole(MW68.08g/mol).

AdjustpHto8.0usingNaOH.Ifthetaggedproteindoesnotbindundertheseconditions,the

amountofimidazoleshouldbereducedto1-5mM.

B.Nativewashbuffer(1L):50mMNaH2PO4;6.90gNaH2PO4·H2O(MW137.99g/mol),300

mMNaCl;17.54gNaCl(MW58.44g/mol),20mMimidazole;1.36gimidazole(MW68.08

g/mol).AdjustpHto8.0usingNaOH.

C.Nativeelutionbuffer(1L):50mMNaH2PO4;6.90gNaH2PO4·H2O(MW137.99g/mol),

300mMNaCl;17.54gNaCl(MW58.44g/mol),250mMimidazole;17.00gimidazole(MW

68.08g/mol).AdjustpHto8.0usingNaOH.

5.IMACresin(e.g.,Ni-NTAAgarose,QIAGEN,Cat.#30210;NiSepharoseHP,GEHealthcare,

Cat.#17-5318-01;orNi-NTAHis•BindResin,Novagen,Cat.#70666-4).

【重组蛋白纯化步骤】

一、重组蛋白的诱导表达

1、将阳性克隆接种至5mL液体LB培养基中，置于摇床中200rpm/37°C培养12h。

Kanamycin（Kan）终浓度为50µg/mL；Ampicillin（Amp）终浓度为100µg/mL。

2、将过夜培养的菌液以1:100接入300mL液体LB培养基中，置于摇床中200rpm/37°C

培养约1.5-2h，测定菌液的OD595值。当OD595达到0.5-0.8时，取1mL菌液，4000×g离

心去除上清，并将沉淀的菌体冻存于−80°C。

3、同时，在剩余的菌液中加入终浓度为0.2mM的IPTG，并继续将菌液