An improved yeast transformation method for the generation of very large human antibody libraries
An improved yeast transformation method for the generation of very large human antibody libraries
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DOI：
10.1093/protein/gzq002
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发表时间：
2010-04-01
影响因子：
2.4
通讯作者：
Hsieh, Chung-Ming
中科院分区：
文献类型：
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作者：
Benatuil, Lorenzo;Perez, Jennifer M.;Hsieh, Chung-Ming
利用酵母展示技术筛选抗体库是一种高效、高灵敏度的筛选靶抗原结合物的方法。然而，由于有限的酵母转化效率，这种强大的选择工具通常受到酵母文库的典型中等大小（类似于10（7））的阻碍，并且用于抗体发现和工程化的酵母展示技术的全部潜力只有在其可以与产生非常大的酵母文库的手段结合时才能实现。我们在这里描述了一种通过电穿孔的酵母转化方法，该方法允许有效地产生大小高达10（10）的大抗体文库。已经测试了多种组分和条件，包括CaCl 2、MgCl 2、蔗糖、山梨糖醇、乙酸锂、二硫苏糖醇、电穿孔电压、DNA输入和细胞体积，以鉴定最佳组合。通过应用该开发的方案，我们基本上在1天内构建了1.4 × 10（10）人脾抗体文库，转化效率为1-1.5 × 10（8）转化体/μ g载体DNA。综上所述，我们已经开发了一种高效的酵母转化方法，该方法能够产生非常大的和多产的人抗体文库用于抗体发现，并且我们现在常规地在一天内制备10（9）个文库用于抗体工程目的。
Antibody library selection by yeast display technology is an efficient and highly sensitive method to identify binders to target antigens. This powerful selection tool, however, is often hampered by the typically modest size of yeast libraries (similar to 10(7)) due to the limited yeast transformation efficiency, and the full potential of the yeast display technology for antibody discovery and engineering can only be realized if it can be coupled with a mean to generate very large yeast libraries. We describe here a yeast transformation method by electroporation that allows for the efficient generation of large antibody libraries up to 10(10) in size. Multiple components and conditions including CaCl2, MgCl2, sucrose, sorbitol, lithium acetate, dithiothreitol, electroporation voltage, DNA input and cell volume have been tested to identify the best combination. By applying this developed protocol, we have constructed a 1.4 x 10(10) human spleen antibody library essentially in 1 day with a transformation efficiency of 1-1.5 x 10(8) transformants/mu g vector DNA. Taken together, we have developed a highly efficient yeast transformation method that enables the generation of very large and productive human antibody libraries for antibody discovery, and we are now routinely making 10(9) libraries in a day for antibody engineering purposes.