将多个蛋白质单体进行有序组装而获得的蛋白质复合物拥有更加复杂的结构和更加优良的功能。相比非共价组装，共价组装蛋白质复合物更加稳定，但已报道的共价组装方法有位阻效应明显、组装效率偏低等缺点。课题组前期研究表明，RIαD/D多肽和AKBI多肽具有分子量小、相互作用强等特点。课题组已经利用该对多肽实现了蛋白质非共价组装。结构分析显示该对多肽结合后，末端距离仅有几个纳米。本研究将利用临近诱导反应原理，设计、表达或合成该对多肽，优化多肽序列和反应条件，建立一种蛋白质共价组装方法。同时，本研究将利用该方法构建治疗慢性粒细胞白血病（CML）的多价抗体类似物（Monobody），通过多价相互作用提高其与Bcr-Abl激酶的SH2结构域的结合能力，解决monobdy固有的与靶分子亲和力低的问题。研究结果将对基于D/D和AKB多肽的正交蛋白质共价组装技术的建立积累宝贵经验，同时为该技术的更广泛应用奠定基础。

Protein complexes constructed from several individual proteins can perform tasks that are beyond the capability of individual proteins. Advancement in bioconjugation technology has opened the door to construct sophisticated protein nanostructures that are of revolutionary value in medicine, synthetic biology and nanotechnology. Although covalently linked multi-protein complex is obviously more stable than that of non-covalently linked one, current strategies for construction of covalently linked multi-protein complexes often interfere with the functionalities of the proteins because of steric hindrance and/or result in low assembly yield. . The specific recognition of A-kinase binding (AKB) domain of A-kinase anchoring proteins (AKAPs) with the dimerization and docking (D/D) domain of regulatory (R) subunits of protein kinase A (PKAs) represents a key regulatory mechanism to precisely localizes the kinase activity at specific locations in the cell. We envision the attractive traits of this interaction, including the unique folding and binding pattern and its dynamicity and specificity, can be translated to a strategy for multi-protein complex construction. In our previous study, by engineering RIα type D/D (RIαD/D) domain and the specific peptide AKBI, we have successfully constructed non-covalent multi-protein complexes.. On the basis of the principle “proximity-induced reactivity”, here we will express (or synthesis) and purify RIαD/D and AKBI with different sequence design. By optimizing the reaction conditions, finally, we will develop a new method for construction of covalently linked multi-protein complex. In view of the relative lower binding affinity between monobody and its target molecule, we will also plan to showcase in this new technology in construction of stably tethered multi-valent monobody that specifically binds with Bcr-Abl kinase SH2 domain. We envision that the multivalent monobody can bind SH2 domain more tightly through multivalent interaction. As a result, the inhibition level of the Bcr-Abl kinase activity will be much higher by using multivalent monobody. . Taken together, the AKBI and RIαD/D peptides lead us to a new platform technology with great extensibility and proven applicability in different fields. We foresee, the success on RIα-D/D based strategy for covalent assembly of multi-proteins can guide the engineering of other D/D domains and the aforementioned application in construction of multivalent monobody is only the tip of the iceberg of this enabling technology.