The intracellular Ig fold: a robust protein scaffold for the engineering of molecular recognition
The intracellular Ig fold: a robust protein scaffold for the engineering of molecular recognition
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DOI：
10.1093/protein/gzs007
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发表时间：
2012-05-01
影响因子：
2.4
通讯作者：
Mayans, Olga
中科院分区：
文献类型：
--
作者：
Bruning, Marc;Barsukov, Igor;Mayans, Olga
支持分子识别的蛋白质支架在生物技术中有多种应用。因此，具有坚固结构核心但具有适应性表面环的蛋白质框架是持续需求的。最近，在细胞内起源的Ig结构域，特别是肌纤丝的模块化成分的表征方面取得了显著进展。这些Ig属于I（中间）型，非常稳定，高可溶性，不需要在大肠杆菌的细胞质中产生。以titin的Z1结构域为代表，我们发现I-Ig折叠可以容忍其CD环的剧烈多样化，构成一个有效的肽显示系统。我们使用差示扫描荧光法、傅里叶变换红外光谱和核磁共振检查了cd环接枝的z1肽嵌合体的稳定性，并证明在该位置引入生物反应性亲和粘合剂不会损害结构域的结构完整性。此外，使用下拉法和等温滴定量热法分析了外源肽序列在Z1中的结合效率。我们发现，一个内部嫁接的、亲和的FLAG标签在折叠环境中起作用，在溶液和亲和凝胶中与抗FLAG M2抗体相互作用。总之，这些数据揭示了细胞内Ig支架靶向功能化的潜力。
Protein scaffolds that support molecular recognition have multiple applications in biotechnology. Thus, protein frames with robust structural cores but adaptable surface loops are in continued demand. Recently, notable progress has been made in the characterization of Ig domains of intracellular originin particular, modular components of the titin myofilament. These Ig belong to the I(intermediate)-type, are remarkably stable, highly soluble and undemanding to produce in the cytoplasm of Escherichia coli. Using the Z1 domain from titin as representative, we show that the I-Ig fold tolerates the drastic diversification of its CD loop, constituting an effective peptide display system. We examine the stability of CD-loop-grafted Z1-peptide chimeras using differential scanning fluorimetry, Fourier transform infrared spectroscopy and nuclear magnetic resonance and demonstrate that the introduction of bioreactive affinity binders in this position does not compromise the structural integrity of the domain. Further, the binding efficiency of the exogenous peptide sequences in Z1 is analyzed using pull-down assays and isothermal titration calorimetry. We show that an internally grafted, affinity FLAG tag is functional within the context of the fold, interacting with the anti-FLAG M2 antibody in solution and in affinity gel. Together, these data reveal the potential of the intracellular Ig scaffold for targeted functionalization.