Development of a high-throughput screen for protein catalysts: application to the directed evolution of antibody aldolases.
Development of a high-throughput screen for protein catalysts: application to the directed evolution of antibody aldolases.
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DOI：
10.1002/anie.200352117
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发表时间：
2003-12
影响因子：
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通讯作者：
J. Gildersleeve;A. Varvak;S. Atwell;Doug Evans;P. Schultz
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文献类型：
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作者：
J. Gildersleeve;A. Varvak;S. Atwell;Doug Evans;P. Schultz
高通量筛选可以是鉴定和进化生物催化剂的有力方法。[1-3]迄今为止，几乎所有的高通量筛选（例如蓝色/白色菌落筛选、平板提升、细胞分选等）测定活细胞或粗细胞裂解物中的催化活性。因此，对可使用的底物、反应和条件存在显著限制。此外，在许多蛋白质、脂质、核酸和其他细胞组分存在下检测产物可能是有问题的。为了克服这些限制，我们开发了一种高通量系统来表达、纯化和测定蛋白质的催化活性。通过对纯化的蛋白质进行检测，可以探索更广泛的底物和反应，包括涉及细胞不可渗透底物、内源性背景活性和非显色产物的底物和反应。为了说明这个系统的潜力，我们已经将其应用到具有醛缩酶活性的催化抗体的定向进化。第一步是开发一个通用系统，用于平行表达和纯化大量抗体。[4]该系统应适合于表达水平和催化活性的优化，因为两者都是抗体开发的重要特性。我们的方法是培养高密度的1 mL的E.大肠杆菌在96孔板中，裂解细胞，然后用Ni-NTA珠捕获His标记的抗体。为了使系统的效用最大化，需要一组能够容纳许多不同抗体的表达和纯化条件。此外，必须产生足够量的纯化抗体以在催化测定中获得良好的信噪比。蛋白质的表达水平和纯度也必须是可再现的，以便可以有信心地检测活性和表达水平具有增量变化的突变体。最后，协议应兼容自动化和高通量的格式，步骤需要大量的操作板，如离心和超声处理被avoided.After广泛的优化，我们开发了一个半自动化系统，能够常规筛选6000克隆/运行在一个平行的96孔格式。蛋白质产量范围为0.5-3 μg/孔（1 mL培养物/孔），纯度大于90%。此外，广泛的抗体已经成功地表达和纯化使用该系统，而无需修改协议。[5]典型的筛选开始于用编码与His 6标签融合的突变抗体（小鼠-人嵌合Fab）的质粒文库转化细菌（Top10）。[6]表达载体是一种经修饰的pBAD质粒，其表达受阿拉伯糖启动子控制。使用机器人菌落采集器将单菌落采集到含有2xYT/amp的浅96孔板的单个威尔斯孔中。将这些起始培养物培养过夜，然后从每个孔中转移10 μL至2 mL深96孔板中相应的1 mL表达培养物中。[7]为了加快这一过程，每个板所需的96个液体转移是使用配有96针头的机器人液体处理器并行完成的。使起始培养物生长至饱和，以确保每种表达培养物以大约相同的密度接种。此外，起子培养物用作筛选期间鉴定的命中物的质粒DNA的来源（起子培养物在48 ℃下储存直至筛选完成）。然后将培养物在37 ℃和250 rpm下生长7 h，并通过平行加入阿拉伯糖（4%，50 μL/孔）诱导蛋白质产生。将培养物在258 ℃和250 rpm下振摇12 h，然后加入100 μL/孔的...
High-throughput screens can be powerful methods for identifying and evolving biological catalysts.[1–3] To date, nearly all high-throughput screens (eg blue/white colony screens, plate lifts, cell sorting, etc.) assay catalytic activity in live cells or crude cell lysates. As a result, there are significant limitations on the substrates, reactions, and conditions that can be used. In addition, detection of products in the presence of many proteins, lipids, nucleic acids, and other cellular components can be problematic. To circumvent these limitations, we have developed a high-throughput system to express, purify, and assay the catalytic activity of proteins. By carrying out assays on purified proteins, a much wider range of substrates and reactions can be explored, including those involving cell-impermeable substrates, endogenous background activities, and nonchromogenic products. To illustrate the potential of this system, we have applied it to the directed evolution of catalytic antibodies with aldolase activity. The first step involved the development of a general system for expressing and purifying large numbers of antibodies in parallel.[4] The system should be amenable to optimization of expression levels and catalytic activity as both are important properties for antibody development. Our approach was to grow high-density 1-mL cultures of E. coli in 96-well plates, lyse the cells, and then capture the His-tagged antibodies with Ni-NTA beads. To maximize the utility of the system, a single set of expression and purification conditions was required that would accommodate many different antibodies. In addition, sufficient quantities of purified antibodies must be produced to obtain good signal-to-noise ratios in catalytic assays. The expression levels and purity of protein must also be reproducible so that mutants with incremental changes in activity and expression levels can be detected with confidence. Finally, protocols should be compatible with automation and high-throughput formats; steps requiring extensive manipulation of plates such as centrifugation and sonication were avoided.After extensive optimization, we developed a semiautomated system capable of routinely screening 6000 clones/run in a parallel 96-well format. Yields of protein range from 0.5–3 μg/well (1 mL culture/well) and purities are greater than 90%. In addition, a wide range of antibodies have been successfully expressed and purified using the system without modifying the protocol.[5] A typical screen starts by transforming bacteria (Top10) with a library of plasmids encoding mutant antibodies (mouse–human chimeric Fabs) fused to His6 tags.[6] The expression vector is a modified pBAD plasmid with expression controlled by the arabinose promoter. Single colonies are picked into individual wells of shallow, 96-well plates containing 2xYT/amp using a robotic colony picker. These starter cultures are grown overnight and then 10 μL is transferred from each well to a corresponding 1 mL expression culture in a 2-mL, deep, 96-well plate.[7] To expedite the process, the 96 liquid transfers required for each plate are done in parallel using a robotic liquid handler fitted with a 96-needle head. The starter cultures are grown to saturation to ensure that each expression culture is inoculated at about the same density. In addition, the starter cultures serve as a source of plasmid DNA for hits identified during the screen (starter cultures are stored at 48C until completion of the screen). The cultures are then grown for 7 h at 378C and 250rpm, and protein production is induced by parallel addition of arabinose (4%, 50 μL/well). Cultures are shaken at 258C and 250 rpm for an additional 12 h, and then 100 μL/well of …