免疫球蛋白可变域框架区域的序列耐受性与非正规的内域二硫化物链接
Dae Young Kim1, Hiba Kandalaft1, Michael J Lowden1
1Life Sciences Division, Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, Ontario, Canada.
The Journal of biological chemistry
|September 24, 2023
概括
在免疫球蛋白可变域中设计的非正规的二硫化物链接增强了稳定性和蛋白酶抵抗性. 这些在人类中缺少的新型链接与抗原结合相容,并改善蛋白质折叠.
科学领域:
- 结构生物学 结构生物学
- 蛋白质工程是指蛋白质工程.
- 免疫学 免疫学 免疫学
背景情况:
- 免疫球蛋白 (Ig) 域通常具有单个保存的二硫化键 (Cys23-Cys104).
- 非正规的二硫化物链接可能会增强Ig域的稳定性.
研究的目的:
- 详尽研究Ig变量 (V) 域框架区域 (FRs) 对非正规的二硫化物链接的序列耐受性.
- 为了确定新的二硫化物链接,以改善V域的稳定性和功能.
主要方法:
- 生成和选的菌体显示图书馆的工程VH域与邻近的β-链的所有配对氨酸组合.
- 引入了已识别的非正规二硫化物链接到不同的VH,VL和VHH域.
- 评估域折叠,稳定性,热稳定性,蛋白酶抵抗性,表达性,可溶性和抗原结合.
主要成果:
- 在VHFR中确定了七种新型的氨酸对,这些氨酸对可以挽救域折叠和稳定性.
- 证明了这些非正规的二硫化物链接的一个子集可以提高热稳定性和蛋白酶抵抗性.
- 证实这些增强没有显著影响表达,溶解性或抗原结合.
结论:
- 在不同的FR位置上,Ig V域对非正规的二硫化物链接表现出意想不到的宽容性.
- 工程非正规的二硫化物键,缺少在人类自然的剧目,提高Ig V域的稳定性和功能.
- 这项研究提供了对Ig V域FRs中工程非正规二硫化物结合的最全面的评估.
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