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[求助]
融合蛋白不挂柱，急！
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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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【答案】应助回帖
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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
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 |
5楼2011-09-02 16:57:40
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