Biologics Department, Abbott Bioresearch Center, Worcester, MA 01605, USA.
Protein Eng Des Sel. 2010 Apr;23(4):155-9. doi: 10.1093/protein/gzq002. Epub 2010 Feb 3.
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 (approximately 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 CaCl(2), MgCl(2), 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/microg 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.
酵母展示技术的抗体库选择是一种高效、高灵敏度的方法，可以鉴定与靶抗原结合的配体。然而，由于酵母转化效率有限，通常较小的酵母文库（约 10^7）限制了该强大选择工具的应用，只有将其与产生非常大的酵母文库的方法结合起来，才能充分发挥酵母展示技术在抗体发现和工程中的潜力。我们在这里描述了一种通过电穿孔进行酵母转化的方法，该方法可有效产生高达 10^10 大小的大型抗体文库。我们已经测试了包括 CaCl2、MgCl2、蔗糖、山梨醇、醋酸锂、二硫苏糖醇、电穿孔电压、DNA 输入和细胞体积在内的多种成分和条件，以确定最佳组合。通过应用该开发的方案，我们在一天内构建了一个 1.4×10^10 的人脾抗体文库，转化率为 1-1.5×10^8 转化子/μg 载体 DNA。总之，我们开发了一种高效的酵母转化方法，可用于产生用于抗体发现的非常大且高产的人抗体文库，我们现在常规每天用于抗体工程目的构建 10^9 文库。
