Bio-protocol
·
2025
·Vol. 15
·期 8
·e5286
·
IF 1.1
Xiaofeng Yang,Zhanglin Lin,Ya Xiang,Zisha Lao
Antibody purification is a fundamental technology for therapeutic and diagnostic applications. While traditional methods like ammonium sulfate precipitation and polyethylene glycol precipitation are cost-effective, they often result in low purity and require multiple purification steps. Protein A–based affinity chromatography, the gold standard for antibody purification, provides high specificity but can be further improved to increase loading capacity and reduce costs. In this protocol, we introduce a novel approach for purifying high-quality, high-purity antibodies from complex samples using SpyFixer/Z domain–modified resin. This method utilizes Spy chemistry for site-specific immobilization of the Z domain of Protein A, significantly enhancing antibody loading capacity up to 200 mg/mL resin and ensuring stable, durable immobilization. Using this protocol, we achieved >90% purity of human immunoglobulin G (hIgG) from diverse sources, including E. coli cell lysates, human serum, and industrial fermentation broth. The SpyFixer/Z domain–modified resin protocol is simple, cost-effective, and offers a robust, scalable solution for efficient antibody purification in bioengineering applications. This immobilization scheme based on Spy chemistry can also be extended to other immunoglobulin-binding proteins, such as Protein G and Protein L, to develop high-efficiency affinity resins. Key features • This protocol builds upon purification methods developed by Lin’s lab [1], providing more detailed steps than the previously published study. • The protocol offers a useful and standardized approach for purifying antibodies and Fc-fusion proteins. • The SpyFixer/Z domain–modified resin is easy to prepare, reusable, and offers a cost-effective solution.
研究领域:
生物技术
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免疫学
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生物工程
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JOURNAL OF CHROMATOGRAPHY A
·
2024
·
IF 3.8
Yuxiang Zhang,Yao Zhang,Luyao Zhang,Yongdong Liu
The use of nanobodies (Nbs) in affinity chromatography for biomacromolecule purification is gaining popularity. However, high-performance Nb-based affinity resins are not readily available, mainly due to the lack of suitable immobilization methods. In this study, we explored an autocatalytic coupling strategy based on the SpyCatcher/SpyTag chemistry to achieve oriented immobilization of Nb ligands. To facilitate this approach, a variant cSpyCatcher003 (cSC003) was coupled onto agarose microspheres, providing a specific attachment site for SpyTagged nanobody ligands. The cSC003 easily purified from Escherichia coli through a two-step procedure, exhibits exceptional alkali resistance and structural recovery capability, highlighting its robustness as a linker in the coupling strategy. To validate the effectiveness of cSC003-derivatized support, we employed VHSA, a nanobody against human serum albumin (HSA), as the model ligand. Notably, the immobilization of SpyTagged VHSA onto the cSC003-derivatized support was achieved with a coupling efficiency of 90 %, significantly higher than that of traditional thiol-based coupling method. This improvement directly correlated to the preservation of the native conformation of nanobodies during the coupling process. In addition, the Spy-immobilized resin demonstrated better performance in the binding capacity, with a 3-fold improvement in capture efficiency, underscoring the advantages of the Spy immobilization strategy for oriented immobilization of VHSA ligands. Moreover, online purification and immobilization of SpyTagged VHSA from crude bacterial lysate was achieved using the cSC003-derivatized support. The resulting resin exhibited high binding specificity towards HSA, yielding a purity above 95 % directly from human serum, and maintained good stability throughout multiple purification cycles. These findings highlight the potential of the Spy immobilization strategy for developing Nb-based affinity chromatographic materials, with significant implications for biopharmaceutical downstream processes.
研究领域:
生物化学
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分子生物学
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生物工程
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JOURNAL OF CHROMATOGRAPHY A
·
2024
·
IF 3.8
Shuxian Zhang,Xiaoying Wang,Xiaofei Wang,Xiaoxuan Fan,Keshuai Liu,Yuping Sa,Gidion Wilson,Xueqin Ma,Guoning Chen
α-Glucosidase plays a direct role in the metabolic pathways of starch and glycogen, any dysfunction in its activity could result in metabolic disease. Concurrently, this enzyme serves as a target for diverse drugs and inhibitors, contributing to the regulation of glucose metabolism in the human body. Here, an integrated analytical method was established to screen inhibitors of α-glucosidase. This step-by-step screening model was accomplished through the biosensing and affinity chromatography techniques. The newly proposed sensing program had a good linear relationship within the enzyme activity range of 0.25 U mL −1 to 1.25 U mL −1 , which can quickly identify active ingredients in complex samples. Then the potential active ingredients can be captured, separated, and identified by an affinity chromatography model. The combination of the two parts was achieved by an immobilized enzyme technology and a microdevice for reaction, and the combination not only ensured efficiency and accuracy for inhibitor screening but also eliminated the occurrence of false positive results in the past. The emodin, with a notable inhibitory effect on α-glucosidase, was successfully screened from five traditional Chinese medicines using this method. The molecular docking results also demonstrated that emodin was well embedded into the active pocket of α-glucosidase. In summary, the strategy provided an efficient method for developing new enzyme inhibitors from natural products.
研究领域:
生物化学
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药物开发
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分析化学
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高影响因子
Nanoscience and Nanotechnology Letters
·
2018
·Vol. 10
·期 3
·435-439
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IF 2.917
Xu Hongpan,Sun Tingting,Huang Yuanfu,Song Lian,Li Zhiyang,Wang Wei
Placing of affinity tags at desired position of the proteins has been widely used to facilitate protein separation. One of the most commonly method for protein separation is nickel ion (Ni2+) affinity chromatography. Complex steps and time consuming in nickel ion affinity
chromatography have caused high demand for alternative purification methods that allow direct and rapid separation of His-tagged proteins. In the present study, magnetic core–shell beads enriched with Ni2+-nitrilotriacetate (Ni-NTA) on their surface were prepared in this study
by precipitation polymerization, with a uniform size of about 10 μm. The presented Ni2+ amount was 2.23%, which endowed the spheres with excellent magnetic responsivity and dispersity. We compared the magnetic beads enrichment method and nickel ion affinity chromatography
method for extraction of His-tagged protein sample with different concentrations. The results revealed that, when compared with nickel ion affinity chromatography, extraction efficiency, purity of the extracted protein, and regeneration effort were much higher with the magnetic beads enrichment
method. By using magnetic beads enrichment method, samples could be separated within 20 s, and purified within 1 h, with unlimited flow velocity and sample capacity. In addition, the magnetic beads enrichment method was particularly promising for the separation of His-tagged proteins in less
samples.
研究领域:
生物化学
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蛋白质组学
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高影响因子
STAR Protocols
·
2023
·
IF 0
Bo Zhou,Ye Chen,Lifang Li,Jiashuo Liu,Yuyan Wang,Zheying Huang,Zheng Hu,Rui Tian,Zhen Li
Summary Here, we present a protocol for generating LtCas12a protein recognizing distinct TTNA (N represented A, T, C, G) protospacer adjacent motif sequence. We describe steps for transforming and harvesting bacterial cells and protein purification including nickel affinity chromatography and dialysis. We then detail procedures for verification of LtCas12a with cis- and trans-cleavage activities. For complete details on the use and execution of this protocol, please refer to Chen et al. (2023). 1
研究领域:
分子生物学
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生物化学
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基因工程
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JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
·
2015
·
IF 2.729
Berlin Zang,Jun Ren,Li Xu,Lingyun Jia
Immobilization of affinity ligands on supporting matrices is a key step for the preparation of affinity chromatography resins, and an efficient coupling strategy can significantly improve the validity and cost of the affinity system, especially for systems that employ expensive recombinant proteins or antibodies as affinity ligands. This study described a simple method for obtaining site-specific immobilization of protein A (the ligand) via aldehyde-hydrazide conjugation and its application in antibody purification via protein A chromatography. An aldehyde group was generated at the N-terminus of protein A in vivo by co-expressing a formylglycine-generating enzyme (FGE) and recombinant protein A containing a FGE recognizing sequence (aldehyde tag) in Escherichia coli . The resulting aldehyde allowed direct immobilization of protein A onto the hydrazide-modified agarose matrices under mild condition. We found that 100 mM aniline was most effective for catalyzing the coupling reaction, and the recombinant protein A could be coupled with high selectivity, directly from a crude cell extract. The site-specific immobilized protein A showed good capacity for antibody purification. The specificity of the aldehyde-hydrazide reaction not only allowed site-specific immobilization of affinity ligands, but also improved the cost of the process by employing unpurified ligands, a method that might be of great use to industrial applications.
研究领域:
生物化学
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分子生物学
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蛋白质工程
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高影响因子
Nature Protocols
·
2013
·Vol. 8
·期 3
·461-480
·
IF 7.96
Zhou Houjiang,Ye Mingliang,Dong Jing,Corradini Eleonora,Cristobal Alba,Heck Albert J R,Zou Hanfa,Mohammed Shabaz
Mass spectrometry (MS)-based proteomics has become the preferred tool for the analysis of protein phosphorylation. To be successful at such an endeavor, there is a requirement for an efficient enrichment of phosphopeptides. This is necessary because of the substoichiometric nature of phosphorylation at a given site and the complexity of the cell. Recently, new alternative materials have emerged that allow excellent and robust enrichment of phosphopeptides. These monodisperse microsphere–based immobilized metal ion affinity chromatography (IMAC) resins incorporate a flexible linker terminated with phosphonate groups that chelate either zirconium or titanium ions. The chelated zirconium or titanium ions bind specifically to phosphopeptides, with an affinity that is similar to that of other widely used metal oxide affinity chromatography materials (typically TiO 2 ). Here we present a detailed protocol for the preparation of monodisperse microsphere–based Ti 4+ -IMAC adsorbents and the subsequent enrichment process. Furthermore, we discuss general pitfalls and crucial steps in the preparation of phosphoproteomics samples before enrichment and, just as importantly, in the subsequent mass spectrometric analysis. Key points such as lysis, preparation of the chromatographic system for analysis and the most appropriate methods for sequencing phosphopeptides are discussed. Bioinformatics analysis specifically relating to site localization is also addressed. Finally, we demonstrate how the protocols provided are appropriate for both single-protein analysis and the screening of entire phosphoproteomes. It takes ∼ 2 weeks to complete the protocol: 1 week to prepare the Ti 4+ -IMAC material, 2 d for sample preparation, 3 d for MS analysis of the enriched sample and 2 d for data analysis.
研究领域:
蛋白质组学
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生物化学
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高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2016
·Vol. 408
·期 27
·7595-7605
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IF 3.125
Bo Chunmiao,Wang Chaozhan,Wei Yinmao
Immobilized metal affinity chromatography (IMAC) has been widely used for the specific separation of biopolymers. However, leakage of metal ions from IMAC adsorbents is of concern in IMAC. In this study, we designed a novel tridenate bis(5-methyltetrazolium)amine (BMTA) to reduce the leakage of metal ions by improving the affinity to immobilized metal ions. The ligand was bonded onto silica via three-step reaction to prepare a high-performance IMAC stationary phase. The chromatographic behaviors of ribonuclease A, cytochrome c , and lysozyme on the Cu(II)-, Ni(II)-, and Zn(II)-chelated stationary phase were investigated with respect to pH effect and elution with an imidazole gradient. The retention times of these three proteins increased by increasing the pH of the mobile phase but decreased by increasing the concentration of the competitive displacer. The retaining strength of the three proteins on the chelated stationary phase were in the order Cu(II) > Ni(II) > Zn(II). The behavior of these three proteins was consistent with the properties of a typical IMAC. The BMTA ligand exhibited a much stronger affinity for Cu(II) and Ni(II) than iminodiacetic acid (IDA), which is often regarded as a standard tridentate IMAC ligand. Quantum mechanical calculations at the B3LYP/6-31G level were used to image the coordination mode of the protein-metal ions-BMTA complex. In addition, a fused histidine-tagged cecropin b-human epidermal growth factor (CB-EGF) from Escherichia coli crude extract was purified by the Ni(II)-chelated stationary phase, and the purity of the CB-EGF was determined to be at least 90 %. These results suggest that the BMTA ligand may have potential applications in the preparation of therapeutics.
A novel ligand of tridenate bis(5-methyltetrazolium)amine (BMTA) was designed to reduce the leakage of metal ions from the column in immobolized metal affinity chromatography (IMAC).
研究领域:
生物化学
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分析化学
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制药工程
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Journal of Materials Chemistry B
·
2016
·Vol. 4
·期 11
·1960-1967
·
IF 4.872
Senwu Li,Kaiguang Yang,Baofeng Zhao,Xiao Li,Lukuan Liu,Yuanbo Chen,Lihua Zhang,Yukui Zhang
Recombinant protein technology occupies an important position in fields including biopharmaceutics, proteomics, structural and functional biology. However, the purification of His-tagged protein, the majority portion of recombinant protein, is seriously hindered by impurities. These impurities, including host proteins with inherent cysteine and histidine-rich regions or metal centers, are usually beyond the purification ability of commonly used IMAC materials. To remove this barrier, a novel purification material was developed through enhancing the selectivity of IMAC by means of surface epitope imprinting using His-tag, the common terminal of His-tagged protein, as the template. Characterizations including TEM, thermogravimetric analysis, X-ray photoelectron spectroscopy, measurement of DLS size and zeta potential were carried out to prove the fabrication of the imprinted shell. Results exhibited a high imprinting factor of 7.1. Besides, the adsorption kinetics were not affected by the surface imprinted shell and could reach adsorption equilibrium within 15 min. Compared with the substrate IMAC, the novel epitope imprinting enhanced IMAC (EI-IMAC) showed an obvious improvement (5% increase of purity) in the selectivity of His-tagged recombinant protein from crude cell lysis.
研究领域:
生物制药
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蛋白质组学
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结构生物学
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功能生物学
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高影响因子
CYTOTECHNOLOGY
·
2011
·Vol. 64
·期 2
·157-163
·
IF 1.277
Yi Yu,Wang Zhuhuan,Li Min,Zhu Keyin,Ying Guoqing
Monoclonal antibodies (McAbs) against chloramphenicol (CAP) were produced to detect CAP residues, which could be toxic and possesses a potential threat to human health. The CAP-BSA conjugate was obtained by bovine serum albumin (BSA) coupled with CAP, and used to immunize the mice. The splenocytes from the immunized mice were fused with mouse myeloma cells SP2/0 to form hybridoma, which may secrete McAbs against CAP. Hybridomas 1D 1 and 3G 12 secreting McAbs against CAP were obtained by screening. Ascites containing McAbs were prepared by injecting 1 x 10 6 cells of hybridoma 1D 1 and 3G 12 into the abdomen of mice. Protein A affinity chromatography was used to purify McAbs against CAP in a single chromatographic step with recovery yield above 80% and purity above 95% and full recovery of antibody activity. Experiments showed that McAb 3G 12 was highly specific for CAP and had no cross-reactivity with analogues which have a structure similar to CAP. The IC 50 value was 50.8 ng/mL.
研究领域:
免疫学
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药理学
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毒理学
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Yu-Ting Li,Jia-Lu Pei,Tong Huan,Yue Wu,Ye-Wang Zhang
Although heparinase II has been widely used in the structural analysis of heparin and HS, it suffers from low activity and inefficient expression and purification. In this study, heparinase II from Mariniphaga anaerophila ( Ma HepII) was cloned, and the GST tag was fused to the backside of the enzyme's active pocket. The fusion protein GST- Ma HepII was efficiently expressed in the recombinant Escherichia coli BL21. Afterward, Ni-NTA affinity chromatography was used to purify GST- Ma HepII, with an enzymatic activity recovery yield of 70.3 % and a specific activity of 7.5 U mg −1 toward heparin. When HS was used as the substrate, the enzyme exhibited a specific activity of 11.1 U mg −1 . Additionally, the influence of the GST tag on the enzymatic properties of Ma HepII was evaluated. After removal of the GST tag by proteolytic cleavage, Ma HepII was purified by GSH affinity chromatography. Characterization of the enzymatic properties indicated that the GST tag improved the catalytic efficiency of Ma HepII by 3.1-fold toward heparin and 1.4-fold toward HS. Molecular docking revealed that the GST tag fusion might increase the flexibility of the protein structure and alter the size or shape of the substrate channel. These changes facilitate substrate access to the active site and enhance the catalytic efficiency of Ma HepII.
研究领域:
酶工程
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分子生物学
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蛋白质表达与纯化
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结构生物学
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生物催化
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ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Xinxin Zheng,Sidi Cun,Jiatai Yin,Xue Zhao,Yunshan Wang,Jing Wang,Qian Li,Xinfeng Zhao
The sensitivity, selectivity, and accuracy of immobilized protein-based methods are critically dependent on the strategies employed for protein immobilization. Compared with random immobilization approaches, site-specific covalent methods have emerged as promising alternatives, offering enhanced analytical performance. However, these methods typically require genetic modification of the target protein to incorporate a specific tag or prior purification of the protein, posing significant challenges for immobilizing endogenous proteins. Herein, we address these limitations by using the 5-hydroxytryptamine transporter (5-HTT) as a model system. We designed a probe by conjugating fluvoxamine─a specific ligand of 5-HTT─with either a fluorescent reporter or aminopropyl-modified silica gel through an alkyl linker containing an acyl imidazole moiety as the reactive group. By introducing ligand-directed acyl imidazole (LDAI) chemistry, we achieved selective labeling of 5-HTT in live cells and endogenous 5-HTT immobilization without purification in advance. Labeling experiments confirmed the high specificity and favorable kinetics of the LDAI ligands toward 5-HTT. Chromatographic analysis revealed significant improvements in selectivity (5.7–50.9-fold), resolution (1.2–3.0-fold), and sensitivity (1.0–12.0-fold) for analyzing canonical ligands when using immobilized endogenous 5-HTT, compared to His-tagged and Halo-tagged recombinant proteins. This approach, by substituting fluvoxamine with other ligands in the probe, can be generalized for the labeling and immobilization of a wide range of endogenous proteins, offering a versatile platform for advanced protein-based analytical techniques.
研究领域:
生物化学
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分析化学
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药物化学
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高影响因子
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