Background: Protein immobilization method play a pivotal role in developing affinity chromatographic assays for both basic and applied research, and site-specific, covalent attachment serves as the most desirable strategy. Traditional strategy often gives rise to inconsistent results due to the use of heterobifunctional cross-linker or a similar two-step protocol. Introducing unnatural amino acids into the immobilization is possible to address the issue, however, it needs purification of the protein whereas suffers from loss of the protein activity. Herein, this work developed a method by integrating a thiol-ene addition click reaction during the protein expression for preparation of immobilized protein.

Results: The methodology involved the genetically incorporated O-allyl-l-tyrosine (O-ALTyr) into serotonin transporter (SERT) and norepinephrine transporter (NET), the fusion proteins were immobilized onto silica gel through " thiol-ene " click reaction. The activities of the immobilized proteins were verified by surface plasmon resonance (SPR) measurement with high success rates of 91.4 % for immobilized SERT and 92.8 % for immobilized NET. The stability and repeatability of immobilized proteins were quantified by determination of protein-drug binding parameters by affinity chromatography under diverse conditions including extreme pHs (pH = 6.0-8.0) and high concentrations of denature reagents (5 %-20 % for DMSO and 5 %-40 % for methanol). Finally, screening analysis found that crocin I and n-butylphthalide were dual-target compounds binding to SERT and NET in Gardenia jasminoides Ellis- Rhizoma Ligustici Chuanxiong Hort (GR) extract. The accuracy of the chromatographic screening method was validated by determined the regulation of the two compounds on release of neurotransmitters including 5-HT, NE, and DA, as well as the expression of the SERT and NET.

Significance: This efficient, purification-free, site-specific, and one-step immobilization method proved to yield immobilized protein with highly enhanced stability and reproducibility which is possible to be used in challenging systems, especially affinity chromatographic analysis using mobile phase containing organic solvents. It benefits analysis of interaction between protein and drugs with low water-solubility, thereby having potential for strengthening the role of immobilized protein-based methods in many fields like drug discovery and fabricating other protein surface to pursue improved assay performance.

Keywords: Affinity chromatography; Neurotransmitter transporters; Protein immobilization; Surface plasmon resonance; Unnatural amino acids.

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背景： 蛋白质固定化方法在亲和色谱分析的开发中起着关键作用，无论是在基础研究还是应用研究方面。位点特异性共价结合是最理想的策略。传统的方法由于使用异双功能交联剂或类似两步协议通常会导致不一致的结果。引入非天然氨基酸到固定化过程是解决这一问题的一种可能方法，然而这种方法需要纯化蛋白质，并且可能会导致蛋白质活性的损失。在此工作中，我们开发了一种在蛋白质表达过程中集成硫醇-烯加成点击反应的方法来制备固定化蛋白。

结果： 该研究涉及将非天然氨基酸O-烯丙基-L-酪氨酸（O-ALTyr）通过基因工程引入血清素转运体（SERT）和去甲肾上腺素转运体（NET），并通过“硫醇-烯”点击反应将其固定在硅胶上。使用表面等离子共振（SPR）测量验证了固定化蛋白质的活性，固定化的SERT的成功率为91.4%，固定化的NET成功率为92.8%。通过亲和色谱法，在极端pH值（pH = 6.0-8.0）以及高浓度变性试剂（DMSO为5%-20%、甲醇为5%-40%）条件下，定量评估了固定化蛋白质的稳定性和重现性。最终筛选分析发现，栀子金花与川芎提取物中存在同时结合SERT和NET的双重靶标化合物——藏红花素I和丁基苯酞。通过确定这两种化合物对神经递质（包括5-HT、NE和DA）释放以及SERT和NET表达的调节作用验证了亲和色谱筛选方法的准确性。

意义： 这种高效的、无需纯化、位点特异性和一步法固定化方法能够制备出具有高度稳定性和重现性的固定化蛋白质，该方法在挑战性系统（特别是使用含有有机溶剂的流动相进行亲和色谱分析）中可能被应用。这种方法有利于研究低水溶性药物与蛋白质之间的相互作用，并且有望加强固定化蛋白基方法在诸如新药发现和制造其他高性能蛋白表面等领域的角色。

关键词： 亲和色谱；神经递质转运体；蛋白质固定化；表面等离子共振；非天然氨基酸。