客戶采用環(huán)氧基磁珠Homo-epoxy偶聯(lián)親和配基在《International Journal of Biological Macromolecules》發(fā)文
客戶采用環(huán)氧基磁珠Homo-epoxy偶聯(lián)親和配基在《International Journal of Biological Macromolecules》發(fā)文
Affinity-assisted covalent self-assembly of PduQ-SpyTag and Nox-SpyCatcher to construct multi-enzyme complexes on the surface of magnetic microsphere modified with chelated Ni2+
Mianxing Luo, Meng Zhang, Changbiao Chi, Guo ChenDepartment of Bioengineering and Biotechnology, Huaqiao University, Jimei Ave. 668, Xiamen 361021, China
Received 8 September 2023, Revised 6 December 2023, Accepted 8 January 2024, Available online 11 January 2024, Version of Record 26 January 2024.
International Journal of Biological Macromolecules
Volume 261, Part 1, March 2024, 129365
https://doi.org/10.1016/j.ijbiomac.2024.129365
Abstract
It is of great significance to study the effect of multi-enzyme aggregation behavior at the interface on the formation of multi-enzyme complexes and their co-catalytic characteristics, which is helpful for us to design and construct immobilized multi-enzyme complex systems for in vitro synthetic biology. Here, a magnetic microsphere with chelated Ni2+, was prepared to explore the self-assembly characteristics of PduQ-SpyTag (P-T) and Nox-SpyCatcher (Nsingle bondC) on its surface, based on the mixed interaction mode consisting the affinity of His-tag/Ni2+ and covalent binding of SpyTag/SpyCatcher. After studying the effect of saturated or unsaturated adsorption of P-T on the covalent binding between P-T and Nsingle bondC at the interface, a possible multienzyme interaction mechanism for the affinity-assisted covalent self-assembly on the Ni2+ chelating surface was proposed. The time evolution of NADH showed that the immobilized P-T/N-C complex formed by this method and the free P-T/N-C complex exhibited similar synergistic catalytic properties, and presented higher catalytic efficiency than the simple mixing of P-T and Nsingle bondC. The optimal catalytic conditions, stability and reusability of the immobilized multi-enzyme complexes prepared in this study were also discussed by comparing them with free enzymes. In this study, we demonstrate a simple and effective strategy for self-assembling SpyTag/SpyCatcher fusion proteins on the surface of magnetic beads, which is inspiring for the construction of more cascade enzyme systems at the interface. It provides a new method for facilitating the rapid construction of immobilized multi-enzyme complexes in vitro from the crude cell lysis.
Keywords
Interfacial self-assemblySpyTag/SpyCatcherMagnetic affinity microsphere
研究界面處多酶聚集行為對多酶復(fù)合物形成及其共催化特性的影響具有重要意義，有助于我們設(shè)計和構(gòu)建體外合成生物學(xué)的固定化多酶復(fù)合物體系。在這里，一個帶有螯合Ni的磁性微球2+，基于His-tag/Ni親和力的混合交互模式，探討了PduQ-SpyTag（P-T）和Nox-SpyCatcher（N單鍵C）在其表面的自組裝特性2+以及 SpyTag/SpyCatcher 的共價結(jié)合。在研究了P-T飽和或不飽和吸附對P-T和N-C單鍵界面共價結(jié)合的影響后，提出了親和力輔助共價自組裝在Ni上可能的多酶相互作用機(jī)制2+ 提出了螯合表面。NADH的時間演化表明，該方法形成的固定化P-T/N-C配合物與游離的P-T/N-C配合物表現(xiàn)出相似的協(xié)同催化性能，催化效率高于P-T和N-C單鍵的簡單混合。通過與游離酶的比較，討論了本研究制備的固定化多酶復(fù)合物的最佳催化條件、穩(wěn)定性和可重復(fù)使用性。在這項研究中，我們展示了一種在磁珠表面自組裝SpyTag/SpyCatcher融合蛋白的簡單有效的策略，這對在界面處構(gòu)建更多的級聯(lián)酶系統(tǒng)具有啟發(fā)性。它提供了一種新方法，用于促進(jìn) 從粗細(xì)胞裂解中快速構(gòu)建體外固定化的多酶復(fù)合物。
關(guān)鍵字：界面自組裝，SpyTag/SpyCatcher，磁性親和微球
2.1. Materials
Epoxy-modified magnetic polystyrene microspheres (4.5 μm) was purchased from PuriMag Biotech (Xiamen, China). Iminodiacetic acid (IDA), nickel sulfate hexahydrate (NiSO4·6H2O), sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) sodium chloride (NaCl), potassium dihydrogen phosphate (KH2PO4), di?potassium hydrogen phosphate (K2HPO4), sodium carbonate anhydrous (Na2CO3), sodium bicarbonate (NaHCO3), glycerol and Ammonium sulfate ((NH4)2SO4) were purchased from Sinopharm Chemical Reagents Company. LB broth, Ni-IDA column, agarose, peptone, yeast extract, kanamycin, β-nicotinamide adenine dinucleotide trihydrate (NAD) and β-nicotinamide adenine dinucleotide disodium salt (NADH) were purchased from Sangon Biotech (Shanghai, China). 1,3-propanediol (1,3-PD) and dithiothreitol (DTT) were purchased from Aladdin. Restriction enzymes and T4 DNA ligase were purchased from Thermo Fisher Scientific. 5 × SDS-PAGE protein sample loading buffer was purchased from Beyotime Biotechnology (Shanghai, China). All of the primers used in this work were synthesized by Genscript (Nanjing, China).+
環(huán)氧基磁珠請參考： 高密度環(huán)氧基磁珠|環(huán)氧基磁性微球（1微米）|PuriMag Homo-Epoxy磁珠-生物磁珠專家 (purimagbead.com)
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