基因直接进化研究进展 —— DNA Shuffling 技术

1. 基因直接进化研究进展——DNA Shuffling技术 吴琦 四川农业大学 二○○三年九月

2. 生物的自然进化 • 进化过程： 突变→自然选择→遗传后代 • 进化结果： 基因多样性：为完成同一功能所表现出的 多个 基因或同一个基因(同源性) 代谢途径的多样性：同样产物，多条途径 代谢产物的多样性：同一底物，不同产物 生物多样性：整个生态系统中的生物

3. 人工获取新基因的方法 • 常规的基因工程方法 生物功能 蛋白质 基因 • 新基因的理性设计和人工合成 根据已有基因的序列和功能进行设计 • 基因的直接进化（directed evolution) 可使已有基因获得新的特性 可获得自然界中不存在的基因 可解决许多新的理论和应用问题

4. 基因直接进化的用途 提高酶活性——天门冬氨酸氨基转移酶活性提高30倍 改变底物特异性和对映异构体选择性—— 酯酶可水解非天然酯类 改善酶的工艺性——冷适应和热适应酶 改变酶的拓扑结构——二体酶变为单体酶 获取许多新的理论知识

5. TLPs 的失活曲线

6. TLP-ste突变蛋白的三维结构

7. 酶拓扑结构的变化

8. 基因直接进化的步骤 • 突变 基因突变库的建立 • 筛选 基因突变库的活体或离体筛选 • 基因复制与遗传

9. 建立基因突变库的方法 • 定向诱变： 点突变——碱基删除、增补和替换 • 随机诱变： 易错PCR法(Error-prone PCR)—— • 降低一种dNTP的量（降至5％-10％） • 加入dITP来代替被减少的dNTP • 缓冲液中另加0.5mmol/L Mn2+

11. 易错PCR

12. 同序法（consensus approach） • 对一组同源蛋白质的氨基酸序列进行比较，以一定的标准程序计算出各氨基酸序列的共有序列； • 人工合成同序基因后重组表达。 Martin Lehmann等（2000～2002）把这种方法应用在真菌植酸酶家族设计合成了同序植酸酶基因，并表达出具热稳定性的同序植酸酶。

13. 真菌植酸酶aa序列的同序比较

14. 同序植酸酶-1的最适温度为71℃，而亲代植酸酶的最适温度为45-55℃，增加了16-26℃, 同序植酸酶-1Tm为78℃，比亲代植酸酶增加了15-22℃，而催化性质与大多数亲本植酸酶相似。

15. DNA Shuffling: • 外显子、单基因和基因家族的重组装 • 随机引物延伸法 • 交错延伸法 • 随机片段活体突变: 线状基因和随机片段共转化酵母细胞

16. 基因突变库的筛选方法 Screening the Library and Selecting the Right Clone • 平板分析 • 使抗生素失效的酶类(如头孢菌素酶，b-乳糖酶） • 提高耐热性 • 易于观察的菌落表型（如菌落颜色等） • 营养缺陷型的辅助筛选

17. 噬菌体展示、细胞表面展示 根据所需要的特性，对经Shuffling后的DNA文库在噬菌体或细菌细胞表面（细菌、纤毛虫细胞表面）的表现特征进行筛选，获得提高目的底物亲和力的突变子。 • 减少筛选工作量 突变文库→大的突变子库→小的突变子库→单克隆

18. 噬菌体展示筛选模式

19. DNA Shuffling技术 DNA改组 DNA洗牌 DNA搅乱重排 DNA Shuffling 1994年，Stemmer等，用DNA Shuffling技术体外快速进化蛋白——有性PCR法 1997年 ，France Aronld研究组将DNA Shuffling技术做了改进——交错延伸法

20. DNA Shuffling的内涵 What is DNA shuffling? • crossovers, • deletions, • insertions, • inversions, • point mutations Darwinian Evolution Natural selection of existing mutations Directed Evolution Targeted selection of created mutations What happens after DNA shuffling • Generation of a large library of novel genes (chimeras) • Selection for improved/desired bio-functions

21. DNA Shuffling的内涵 DNA Shuffling：指DNA分子的体外重组，是基因在分子水平上进行有性重组(Sexual Recombination)。通过改变单个基因(或基因家族，gene family)原有的核苷酸序列，创造新基因，并赋予表达产物以新功能。

22. DNA Shuffling与常规定向进化的比较

23. How DNA shuffling works - 1 • How DNA shuffling is done in the tube • Random fragmentation of a pool of related genes; • Self-priming polymerase reaction and template switching (causing crossovers); • PCR amplification with primers of reassembled products

24. . . . . . . . . . . . How DNA shuffling works - 2 Family gene shuffling library of chimeras Single gene shuffling library of point mutants Similar mutants generated by error-prone PCR, random and site-directed mutagenesis Generating chimeras with crossovers of large blocks of sequences

25. How DNA shuffling works - 3 Fragment Reassemble Select best with DNAseI fragments recombinants X XXX X XXX X XXX XX X XX X XX X XXX X XXX X XXX X XX XXX X X X X X XX X X XX X X XX X X XX X XX X XX X XX X Repeat for multiple cycles

26. DNA 重组装的过程

27. 随机引物PCR和重组装

29. 交错延伸PCR突变法

30. DNA Shuffling技术的应用 磷脂酶热稳定－催化活性的分子进化（Jae Kwang Song等，2000）

31. 枯草杆菌蛋白酶E热稳定性的分子进化（Huimin Zhao等，1999）

32. 正面 反面 进化后的枯草杆菌蛋白酶E

33. 耐热p-硝基苯酯酶的分子进化(Lori Giver等，1998）

34. β-葡糖苷酶耐热性的提高 (Marý´a Jesu´ s Arrizubieta等，2000）

35. Evolution of a cytokine using DNA family shuffling Chia-Chun J. Chang ……. Phillip Patten Nature Biotechnology Vol. 17, August, 1999 Aims of the work: Demonstration of usefulness of the DNA shuffling tool for rapidly evolving human a interferon towards improving antiviral property

36. Interferons (IFNa, IFNb, IFNg) • IFNas are produced by monocyte, macrophages and other cells upon stimulation by virus. • IFNs are able to protect cells from viral infection by reacting with IFN receptors on the cell which triggers a series of signal transduction events, hence immune response. • IFNs suppress an exaggerated growth potential of cancer cells. • IFNs can be used as drugs in the treatment of viral infection. • Only one natural IFN-a, Hu-IFN-a2, in clinical studies. • Most active engineered IFN-a, alfacon-1, is a consensus of 13 wild type Hu-IFN-a genes that is used in hepatitis C therapy. • Dose-limiting toxicity, • Receptor cross-reactivity, • Short serum half-lives Reasons for their limited use as drugs:

37. Methods and Materials-1 • DNA Shuffling • Construction of 1st round library: • 8 cloned Hu-IFN genes were shuffled and assembled insert • cloned in the phagemid display vector • Construction of 2nd round 5 libraries: • IFN-CH1. 1xIFN-CH1. 2; IFN-CH1. 1x IFN-CH1.3; • IFN-CH1.1xIFN-CH1. 4;IFN-CH1. 2x IFN-CH1.3; • IFN-CH1.1xIFN-CH1.2x IFN-CH1.3x IFN-CH1.4 (mating of 4 genes). Pair-wise matings

38. Phagemid display of IFN • High-throughput phagemidpreps (Q-BOT colony picker) • Antiviral assays: Cytopathic effect reduction assay on mouse L929 cells, challenging virus-EMCV, 96 well plate format, neutral red, cell fixing, colour extracting, spec OD 540 vs IFNa concentration.

39. Methods and Materials-2 Deconvolution of libraries - screening strategy PEG precip CsCl banded Expression induced phagemids aggregated phagemids E.coli Shuffled products Thousands of single clones picked by robot, placed in 16x96 well plates Expression induced, phagemids aggregated packaged, released 16 pools 96 assayed Library intro- into E.coli All the 36 clones in the 3 pools were prepared, purified & assayed Active pool broken into 8 pools of 12 All 8 pools assayed with 3 having activity So 16 + 8 + 36 = 60 assays were carried out instead of 16 x 96 = 1536

40. Methods and Materials-3 • CHO (Chinese Hamster Ovary) expression and purification(His tag) • of chimeric IFN-as. • High-throughput proliferation assays (inhibition of proliferation) in mouse L929 cells: • Standard [3H]thymidine incorporation methods--IFN-a samples • were mixed with L929 cells in 96 well plates, incubated and [H3] • thymidine added to each well. Plates were harvested on Harvester-96 • and thymidine incorporation was counted on a b counter • Human Daudi cell proliferation assays: • 4 most active chimeras and Hu-IFN-a2a were expressed, purified • and assayed for anti-proliferation activity on human Daudi cells

41. Results-1: structure of shuffled IFNas B: The sequence of one of the cycle 2 chimeras, IFN-CH2.2, is aligned with the most potent human and mouse IFN-as. The IFN-a residues that putatively contact the IFN-a receptor are boxed. Residues in Hu-IFN-a1 that have been shown by site-directed mutagenesis to contribute to activity on mouse cells are shaded.

42. Results-2: Activity of parental and shuffled IFN-as in murine cells

43. Results-Anti viral activity ranking Antiviral activity

44. Results-Potency of IFN-as OD540nm (cell viability) Log10[IFN-a] (mg/ml)

45. Results-continued Structural modelling of IFN-CH2.2 based on the NRM structure of Hu-IFN-a 2a. The protein backbone is coloured to indicate the native Hu-IFN-a segments (5 Hu-IFN-as from which it is derived）. The side chains of putative murine IFN-a receptor contacting residues Lys121 and Arg125 are shown

46. Conclusions 1. Unlike site-directed mutagenesis and other protein engineering methods which produce mainly point mutations, DNA family shuffling can generate recombinants with blocks of sequences from more than two parental genes, hence enhancing genetic diversity. 2. DNA family shuffling of Human-IFN-a genes generates recombinants with potent IFN-a activity on a distantly related species (mouse). 3. Evolution of commercial genes and proteins may be practical, even when very complex, time- consuming, or expensive assays are required.

47. 部分DNA Shuffling技术的研究成果

48. 分子进化工程的应用 • 单基因的定向改造 • 序列相关基因的重排 • 生物代谢途径的改造 • 对整个基因组的改造 • 药物的筛选

49. 谢谢！