Hot:来自Ni-NTA的最新数据[创新技巧]

说起6×His标签蛋白的纯化产品，就不得不提到QIAGEN公司的Ni-NTA，Ni-NTA作为6×His标签蛋白纯化的金标准，20多年来已经被从事蛋白表达纯化研究的老师和同学所认可。2007年QIAGEN在生物通上对从事重组蛋白研究的用户进行了问卷调查。结果发现实验者对纯化试剂最关注的两个因素是：纯度和结合量。

如果说你最关注的是目标蛋白的纯度，那么使用Ni-NTA绝对是上上之选。原因在于Ni-NTA中Ni2+是有4个共价键与NTA结合（见图1），这种强结合力使得Ni-NTA比起其它的产品如Ni-IDA更强壮，Ni2+脱落率最低（见图2）。

图1 The extra coordination site (arrowed) in NTA binds nickel ion more tightly than IDA (the ligand used in many competitor resins).The tighter binding means less nickel leaching and provides purer proteins.

图2. An independent study shows that Ni-NTA loses less nickel than any other tested resin. Fifty column volumes of buffer containing various additives was passed through a small column containing 100 μl resin from QIAGEN, Supplier G, S, or I. The flowthrough was pooled and a sample sent for analysis by inductively coupled plasma mass spectrometry (ICP-MS) at Dr.Weßling Laboratories, Bochum, Germany according to DIN EN ISO 17025.Native buffer: 50 mM Na phosphate; 300 mM NaCl; 10 mM imidazole, pH 8.0. Denaturing buffer: 100 mM Na phosphate; 10 mM Tris•Cl; 8 M urea.

很多人可能会问Ni2+脱落会产生什么后果呢？最直接的后果就是造成目标蛋白纯度很低，Ni2+脱落后空出的位点会结合更多的杂蛋白，另外Ni2+脱落率高意味着需要更经常地对Ni柱进行再生，浪费时间和金钱不说，也非常的不环保（产生很多的Ni废液，污染环境）。

说了那么多Ni-NTA与Ni-IDA的区别，我们还是来看具体的实验对比数据吧。利用Ni-IDA和Ni-NTA对表1中的13种6His标签蛋白进行了纯化，结果表明使用Ni-NTA可以获得更高的蛋白纯度。具体对比数据的胶图可以通过点击相应链接获得。

表1 Proteins expressed and purified in the comparison experiments

GenBank accession number	Gene*	Class†	MW (kDa)	Official full name
NM_002648	PIM1 (view gel)	PK	37.2	pim-1 oncogene
NM_006875	PIM2 (view gel)	PK	35.7	pim-2 oncogene
NM_001001852	PIM3 (view gel)	PK	37.4	pim-3 oncogene
NM_003668	MAPKAPK5 (view gel)	PK	55.5	mitogen-activated protein kinase-activated protein kinase 5
NM_002037	FYN (view gel)	PK	62.3	FYN oncogene related to SRC, FGR, YES
NM_001315	p38a (view gel)	PK	43.0	mitogen-activated protein kinase 14
NM_002750	JNK1 (view gel)	PK	45.7	mitogen-activated protein kinase 8
NM_003403	YY1 (view gel)	TF	46.3	YY1 transcription factor
NM_020529	NFKB1A (view gel)	TF	37.1	nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha
NM_005901	SMAD2 (view gel)	TF	53.8	SMAD family member 2
NM_005238	ETS1 (view gel)	TF	51.9	v-ets erythroblastosis virus E26 oncogene homolog 1 (avian)
NM_006331	EMG1 (view gel)	RB	28.2	EMG1 nucleolar protein homolog (S. cerevisiae)
NM_139071	SMARCD1 (view gel)	RB	51.5	SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 1

* Click on the gene name to see the purification comparison gel. CL: Cleared lysate. Each set of three lanes shows from left to right: flow-through, wash, and elution fractions. † PK protein kinase; TF: transcription factor; RB: ribosome binding protein

受关注的另一个主要因素就是纯化试剂的结合量，说到这里，就有一个好消息要和大家一起分享啦：QIAGEN公司近年来通过对Ni-NTA生产流程的优化，使得Ni-NTA对His标签蛋白的结合量可高达50mg/ml树脂。最近QIAGEN在研发QIAgene预制蛋白表达载体的过程中（该产品信息请参考生物通的新品推荐http://www.ebiotrade.com/newsf/2008-5/2008513161005.htm 或点击http://www1.qiagen.com/Products/QIAgenesExpressionKits-Ecoli.aspx），对Ni-NTA 的结合量也进行了一次大规模的实验研究。实验考察了Ni-NTA对来自不同蛋白家族的24种人类蛋白的结合能力。部分数据如图3所示。从实验结果来看，Ni-NTA对His标签蛋白的结合量可高达50-60mg/ml。因此，对于Ni-NTA Superflow或Ni-NTA Agarose来说，它们的结合量均高达50mg/ml (见表2).

图3 Binding of various His-tagged proteins to Ni-NTA. Binding was performed in batch procedures and proteins quantified using the Bradford method.

表2 New protein binding capacity of QIAGEN Ni-NTA matrices

产品名称	结合量
Ni-NTA Superflow	Up to 50 mg His-tagged protein/ml resin
Ni-NTA Agarose	Up to 50 mg His-tagged protein/ml resin
Ni-NTA Magnetic Beads	up to 2 µg His-tagged protein/µl bead suspension

所以说，Ni-NTA一步纯化就能获得高产量高纯度的6×His标签蛋白。那么这对实验者来说意味着什么呢？高纯度意味着通常省去了多次纯化的麻烦，而高产量意味着每次制备的成本很低，为我们省了不少来之不易的课题经费和宝贵的实验时间。这样就可以空下来多读paper，多做实验设计。

说到这里，不能不提Ni-NTA对各种化学试剂的兼容性。实际上，我们需要利用各种化学试剂对纯化的流程进行优化（如NaCl, DTT等）。Ni-NTA能在多种化学试剂的存在下进行标签蛋白的纯化。 这一特点使得我们可以放心的使用各种化学试剂对纯化过程进行优化。举个例子来说在市场上所有的6×His标签蛋白纯化试剂中，只有Ni-NTA可以兼容10 mM DTT. 点击以下链接，浏览Ni-NTA在化学试剂兼容性方面的最新数据full list of reagents compatible with Ni-NTA.

图4 First and second elution fractions of 6xHis-tagged HIV r everse transcriptase purified using Ni-NTA Superflow under native conditions in buffers containing the indicated concentration of DTT. Results from RT-PCR experiments performed with 6xHis-HIV-RT purified using the indicated concentration of DTT. A 1.7 kb fragment from the human -actin gene was reverse transcribed using 50 ng total RNA, a dT15 primer, and 100 ng of 6xHis-HIV-RT for 1 h at 37°C. 1/20 of the RT reaction was transferred to a PCR reaction using QIAGEN® Taq DNA Polymerase. C: control, 4 units Omniscript® Reverse Transcriptase. NTC: no template control. M: markers.

最后需要指出的是QIAGEN的Ni-NTA产品种类齐全，从ng级到生产级别（kg）都有相应的产品提供（见下图），根据手中要纯化的蛋白的数量和特点选择合适的Ni-NTA产品是我们迈向纯化实验成功的第一步。