框架和互补性决定区域H1负荷对人类VH-B1a域表达,折叠,稳定性和可溶性的影响
Sharmin Sultana1, C Ronald Geyer2
1Department of Biochemistry, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada.
通过引入带电氨基酸来改善稳定性和可溶性来设计可变重链 (VH) 域. 带正电荷的框架区域增强了温度稳定性,而CDRH1的修改对中性和带正电荷的域的影响化温度和溶解度不同.
科学领域:
- 生物化学 生物化学
- 蛋白质工程是指蛋白质工程.
- 免疫技术是一种免疫技术.
背景情况:
- 单域抗体,如可变重链 (VH) 域,在诊断和治疗中比传统抗体具有优势.
- 然而,由于补充性决定区域 (CDR) 中的疏水性残留物,VH域可以聚集,可能导致免疫反应.
- 提高VH域稳定性和溶解性对于它们的治疗应用至关重要.
研究的目的:
- 为增强稳定性和可溶性设计人类的VH-1Ba域.
- 研究在框架区域和CDRH1中引入带电氨基酸对VH域属性的影响.
- 确定设计具有改进生物物理特征的VH域的策略.
主要方法:
- 引入带电氨基酸到人类VH-1Ba域的框架区域.
- 修改了带电氨基酸的近中性 (VH-B1a) 和正电荷 (+VH) 域的CDRH1循环.
- 评估了重新折叠的能力,稳定性 (热稳定性,化温度 - Tm) 和在室温下溶解性.
主要成果:
- 在VH-1Ba框架中的正电荷增加了热稳定性,但稍微减少了重新折叠和可溶性.
- 负电荷的框架域 (-VH) 不能正确折叠.
- CDRH1的修改影响了Tm和可溶性;中性和轻微阳性CDRH1净电荷改善了VH-B1a的可溶性.
- +VH域在所有CDRH1净电荷中显示出更高的Tms,并且对正电荷增加有更好的耐受性.
- 在CDRH1中特定的素替代促进了聚合,这种效应被+VH域部分减轻.
结论:
- 工程带电氨基酸进入VH域可以调节它们的生物物理性质.
- 框架电荷工程为增强热稳定性提供了一条途径.
- CDRH1修改提供了一种微调溶解度和Tm的手段,这对聚合有影响.
- 具有特定CDRH1特征的+VH域对需要正面电荷的应用具有前景.
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