昆克尔的超大菌体显示图书馆的建造 菌体变异和滚动圆放大
Renhua R Huang1, Michael Kierny2, Veronica Volgina3
1MacroGenics, Rockville, MD, USA. huangr@macrogenics.com.
Methods in molecular biology (Clifton, N.J.)
|September 7, 2023
概括
我们使用Kunkel突变和滚动圆放大 (RCA) 开发了一个大型菌体显示库. 这种方法有效地产生了针对各种蛋白质标的高亲和性单体,包括SARS-CoV-2 RBD.
科学领域:
- 分子生物学分子生物学
- 生物技术是生物技术.
- 蛋白质工程是指蛋白质工程.
背景情况:
- 菌体显示是产生重组亲和反应物的关键技术.
- 菌体显示的成功在很大程度上取决于抗体库的规模和多样性.
研究的目的:
- 描述一种用于构建一个大而多样化的菌体显示库的新方法.
- 用这个库来证明高亲和度单体对多个蛋白质标的隔离.
主要方法:
- 昆克尔突变发生的应用,用于引入遗传多样性.
- 利用滚动圆放大器 (RCA) 进行高效的图书馆放大.
- 构建一个1.1 × 10^11成员库,最小的电孔 (26).
主要成果:
- 成功创建了一个规模和多样性的图书馆.
- 孤立的单体,对多个蛋白质标具有低至亚纳米系的亲和力.
- 目标蛋白包括人类COP9信号酶子单元5 (COPS5),HIV-1 Rev. 与蛋白质相似的蛋白质 (HRBL),Ku70/80异构体,SARS-CoV-2 RBD和TGF-β1.1结合.
结论:
- 与RCA结合的Kunkel突变发生是一种有效的策略,用于构建大型菌体显示库.
- 这种方法可以快速分离各种蛋白质标的高亲和度结合剂.
- 产生的单体在研究和诊断方面具有潜在的应用,包括对SARS-CoV-2的应用.
相关概念视频
Cytoskeletal Proteins in Bacteria
Bacterial cells were initially considered simple, randomly organized structures lacking a cytoskeleton. However, the discovery of cytoskeleton homologs in bacteria led to the change of this opinion. Bacterial cytoskeletal filaments regulate the cell shape, cell polarity, cell division, and partitioning of plasmids during cell division. It was later discovered that bacterial cytoskeletal proteins, mainly actin and tubulin homologs, are diverse compared to their eukaryotic counterparts. On the...
Studying the Cytoskeleton
The cytoskeletal architecture can be studied using different microscopic and biochemical techniques. Electron microscopy was instrumental in discovering the cytoskeletal architecture around the 1960s, which allowed obtaining structural information at a high-resolution level. However, the sample preparation procedure often limits this ability in biological samples. Several protocols have been developed over the years to optimize sample preparation. In one of the protocols known as rotary...
Assembly of Complex Microtubule Structures
Complex microtubule structures are present in resting cells and in dividing cells. In resting cells, they are responsible for maintaining the cellular architecture, tracks for intracellular transport, positioning of organelles, assembly of cilia and flagella. They mediate the bipolar spindle assembly for chromosomal segregation and positioning of the cell division plate in dividing cells. The formation of microtubule complex structures depends on the cell type, cell stage, and cell function.


