Integrating click chemistry into protein expression for efficient preparation of immobilized protein bio-surface with enhanced stability and reproducibility in affinity chromatography.

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.