利用家蚕几丁质结合蛋白CPAP3作为蛋白标签纯化重组蛋白

Purification of recombinant proteins with silkworm chitin-binding protein CPAP3 as a protein tag

摘要:

昆虫中存在多种几丁质结合蛋白。其中，含有ChtBD2几丁质结合域的CPAP3蛋白家族是昆虫表皮中含量最为丰富的蛋白质种类之一，本研究旨在以昆虫几丁质结合蛋白为标签蛋白，探讨其在蛋白纯化领域的应用潜力，同时探究CPAP3蛋白结构域数量与其结合能力的关系。利用家蚕CPAP3-A蛋白(简称CPAP3)作为标签蛋白，以绿色荧光蛋白EGFP为目标蛋白，构建融合表达体系实现二者融合表达。以几丁质/壳聚糖材料作为固定相，通过结合实验验证融合蛋白的结合能力。结果显示，融合蛋白CPAP3-EGFP与4种不同的几丁质/壳聚糖均能发生结合。正交实验表明最佳结合条件为β-几丁质、结合缓冲液pH值6.5，此时吸附率高达70.44%左右。为进一步探究结构域数量与结合能力的关系，对CPAP3蛋白进行了截短处理。结果表明，含有3个结构域的蛋白结合能力显著高于其他2个截短组，说明蛋白标签的结合能力与结构域数量呈正相关。为了有效释放结合在几丁质上的目标EGFP蛋白，在目的蛋白与标签蛋白之间引入了TEV蛋白酶切序列。实验结果显示，TEV蛋白酶酶切后，蛋白在28 kDa处出现了新的条带，证实融合蛋白能够被TEV酶切开，目标蛋白得以释放至溶液上清中。本研究为昆虫几丁质结合蛋白作为亲和标签的应用提供了参考。

Abstract:

There are various chitin-binding proteins in insects. Among them, the CPAP3 protein family containing the chitin-binding domain ChtBD2 is one of the most abundant types in the epidermis. This study aims to use insect chitin-binding proteins as tag proteins to explore their application potential in the field of protein purification, and simultaneously investigate the relationship between the number of domains of CPAP3 protein and its binding capacity. CPAP3-A (abbreviated as CPAP3) from silkworm was used as the tag protein, and enhanced green fluorescent protein (EGFP) as the target protein, to achieve the fusion expression of the insect epidermal protein and EGFP. With chitin/chitosan materials as the stationary phase, the application potential of CPAP3 in protein purification was explored through binding experiments. The results showed that the fusion protein CPAP3-EGFP could bind to four different types of chitin/chitosan. Orthogonal experiment analysis optimized the reaction conditions as follows: β-chitin as the matrix and binding buffer at pH 6.5, under which the adsorption rate reached 70.44%. To further investigate the relationship between the number of domains and binding capacity, we conducted truncation treatment on CPAP3. The results indicated that the protein containing three domains had significantly higher binding capacity than the other two truncated groups, suggesting a positive correlation between the binding capacity of the protein tag and the number of domains. To effectively release the target EGFP bound to chitin, we introduced a TEV protease cleavage site between the target protein and the tag protein. Experimental results showed that after digestion with TEV protease, the protein sample exhibited a new band at 28 kDa, confirming that the fusion protein could be cleaved by TEV protease and the target protein was released into the supernatant. This study provides references for the application of insect chitin-binding proteins as affinity tags.