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瞬时 AID 表达用于原位诱变，提高细胞适应性。

Transient AID expression for in situ mutagenesis with improved cellular fitness.

机构信息

Taiwan International Graduate Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, Taiwan.

Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.

出版信息

Sci Rep. 2018 Jun 20;8(1):9413. doi: 10.1038/s41598-018-27717-2.

DOI:10.1038/s41598-018-27717-2
PMID:29925928
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6010430/
Abstract

Activation induced cytidine deaminase (AID) in germinal center B cells introduces somatic DNA mutations in transcribed immunoglobulin genes to increase antibody diversity. Ectopic expression of AID coupled with selection has been successfully employed to develop proteins with desirable properties. However, this process is laborious and time consuming because many rounds of selection are typically required to isolate the target proteins. AID expression can also adversely affect cell viability due to off target mutagenesis. Here we compared stable and transient expression of AID mutants with different catalytic activities to determine conditions for maximum accumulation of mutations with minimal toxicity. We find that transient (3-5 days) expression of an AID upmutant in the presence of selection pressure could induce a high rate of mutagenesis in reporter genes without affecting cells growth and expansion. Our findings may help improve protein evolution by ectopic expression of AID and other enzymes that can induce DNA mutations.

摘要

激活诱导胞嘧啶脱氨酶（AID）在生发中心 B 细胞中引入转录免疫球蛋白基因的体细胞 DNA 突变，以增加抗体多样性。异位表达 AID 并结合选择已成功用于开发具有理想特性的蛋白质。然而，这个过程很费力且耗时，因为通常需要多轮选择才能分离出目标蛋白质。由于脱靶突变，AID 的表达也会对细胞活力产生不利影响。在这里，我们比较了具有不同催化活性的 AID 突变体的稳定和瞬时表达，以确定在最小毒性下最大积累突变的条件。我们发现，在选择压力下，瞬时（3-5 天）表达 AID 上突变体可以在不影响细胞生长和扩增的情况下，在报告基因中诱导高突变率。我们的发现可能有助于通过异位表达 AID 和其他可以诱导 DNA 突变的酶来改进蛋白质进化。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/865f/6010430/d2f9f5386948/41598_2018_27717_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/865f/6010430/03a8db54775a/41598_2018_27717_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/865f/6010430/ada4db73ca63/41598_2018_27717_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/865f/6010430/680c33f6435f/41598_2018_27717_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/865f/6010430/582f9279d39d/41598_2018_27717_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/865f/6010430/ef5e5d9250cd/41598_2018_27717_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/865f/6010430/d2f9f5386948/41598_2018_27717_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/865f/6010430/03a8db54775a/41598_2018_27717_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/865f/6010430/ada4db73ca63/41598_2018_27717_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/865f/6010430/680c33f6435f/41598_2018_27717_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/865f/6010430/582f9279d39d/41598_2018_27717_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/865f/6010430/ef5e5d9250cd/41598_2018_27717_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/865f/6010430/d2f9f5386948/41598_2018_27717_Fig6_HTML.jpg

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