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小木虫论坛-学术科研互动平台 » 生物医药区 » 分子生物 » 综合其他 » 融合蛋白不挂柱，急！
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甜酒慢品

金虫 (初入文坛)

小木虫

[求助] 融合蛋白不挂柱，急！

大家好，我最近实验遇到了一个问题要请教大家。
我做的是一个膜蛋白的异源表达，载体用的是Bio rad的pPAL7载体，这个载体有一个特色，就是它的tag在我的蛋白质的N端，一旦elution buffer中有氯离子则tag和我的目的蛋白会自动切开。我们通过这个Tag同柱子的亲和层析纯化我的目的蛋白，然后再加入含有氯离子的溶液得到我的目的蛋白。
tag等电点9.0大小8.3kd，目的蛋白等电点11.7大小5.7kd，融合蛋白等电点9.64（软件预测），wash buffer是磷酸钠溶液 pH7.2，融合蛋白在包涵体和可溶性部分均有表达。我每次都摇200ml的菌提取蛋白，可是每次做亲和层析都发现与柱子不结合（可溶性部分和包涵体都试过），望大家指点迷津啊！
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还是那个我，简单，哀愁，幸福，疲惫。。。
1楼 2011-09-02 14:01:18
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辉辉集团

银虫 (正式写手)
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3楼: Originally posted by 甜酒慢品 at 2011-09-02 14:52:31:
你好，我用的是普通的旋转柱做的，只要加溶液进去就行了。

啊 这么高级啊 我有点搞不懂了
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4楼2011-09-02 15:27:36
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辉辉集团

银虫 (正式写手)

【答案】应助回帖

甜酒慢品(金币+1): 谢谢啊！ 2011-09-05 10:08:35
甜酒慢品(金币+1): 2011-09-14 10:35:00
你的上柱条件是什么啊？
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2楼2011-09-02 14:45:15
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甜酒慢品

金虫 (初入文坛)

小木虫
引用回帖:
2楼: Originally posted by 辉辉集团 at 2011-09-02 14:45:15:
你的上柱条件是什么啊？

你好，我用的是普通的旋转柱做的，只要加溶液进去就行了。
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还是那个我，简单，哀愁，幸福，疲惫。。。
3楼2011-09-02 14:52:31
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labrat

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★ ★ ★ ★ ★
dhd997(金币+5): good 2011-09-02 18:21:07
甜酒慢品(金币+5): thank you！ 2011-09-05 10:09:14
hi, i don't really understand chinese , but this is the technical guide generally used for your application:
1. Premature elution of cleaved, target protein is observed:
Fusion protein: Insert Thr-Ser spacer between Profinity eXact
™ tag and target protein by cloning into pPAL7’s SpeI site

Lysis:  For maximum yields, do not use lysis buffers and
reagents that contain triggering ions, specifically Cl– or F–
*Maintain lysate at 4°C prior to loading
*Shorten incubation time
*Utilize lysis buffer with pH <7.0
Wash: Pre-chill wash buffer to 4°C prior to use
*Do not use lysis buffers and reagents that contain triggering ions, specifically Cl–
or F–. When preparing wash buffer, substitute sodium acetate (NaOAc) for NaCl; do not use HCl to adjust pH—phosphoric or acetic acid should be used
• Utilize wash buffer with pH <7.0

2. Target protein remains uncleaved and/or does not elute completely:
Fusion protein: Verify that first amino acid (P1') downstream of the
Profinity eXact cleavage site is not proline
• If first amino acid of target protein is undesirable for cleavage, insert amino acid spacer (e.g., Thr-Ser) immediately downstream of cleavage site by site-directed mutagenesis or clone into SpeI site of pPAL7

Elution: • Incubate resin with elution buffer for longer time (try up to 1 hour at room temperature or overnight at 4°C)
• If performing elution at 4°C, incubate overnight
• Increase fluoride concentration in elution buffer
• Substitute 10 mM sodium azide for fluoride in
elution buffer
• Elute with application of second volume of elution
buffer to spin column resin

3. Eluate contains significant contaminants:
Load:• Dilute lysate for multimeric proteins
• Reduce fusion protein load amount to achieve subsaturating level bound to resin
• Incubate lysate with resin for up to an hour at 4°C to increase binding capacity of target fusion protein

Wash: • Perform additional wash step
• Reduce nonspecific, electrostatic binding by increasing ionic concentration of wash buffer; use up to 0.3 M Sodium phosphate, NaOAc, or (NH4)2SO4 (pH 7.2), or amend wash buffer with any of the aforementioned salts. Do not use NaCl
• Reduce hydrophobic interactions by decreasing salt concentration of wash buffer
• Supplement wash buffer with suitable detergent

4. Fusion protein binds poorly to the resin:
Fusion Protein : Proteins with high N-terminal structure may interfere with binding; adding amino acid spacer (i.e., Thr- Ser) between Profinity eXact tag and target protein will improve tag’s accessibility

Lysis : • If using urea to help solubilize proteins, use [urea] <4 M; higher urea concentrations reduce binding capacity

Load : • Allow lysate to incubate with resin for up to an hour at 4°C or for 30 minutes at room temperature; lysates with fusion proteins >75 kD often benefit
from a longer incubation period
• When purifying multimeric proteins, avoid saturating resin; diluting lysate may also improve yields

Resin :• Resin previously used must be regenerated to remove cleaved Profinity eXact tag; apply three column volumes of 0.1M H3PO4 to resin
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5楼2011-09-02 16:57:40
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