JOURNAL OF CHROMATOGRAPHY A
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2024
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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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PubMed
高影响因子
Molecular Catalysis
·
2021
·
IF 5.062
Fei Peng,Qing-Sheng Chen,Min-Hua Zong,Wen-Yong Lou
Sequential immobilization of multiple enzymes can not merely improve the enzymatic activity and enzyme stability but also facilitate the recovery for reuse, well imitating the natural enzyme complexes. Here, we developed a novel approach of preparing sequential multienzyme nanomachineries based on chemical crosslinking and protein assembly, and then characterized the nanodevice. In the preparation process, Kg BDH-SC (SpyCatcher-fused (2 R ,3 R )-2,3-butanediol hydrogenase) was firstly immobilized onto the functionalized silicon dioxide nanoparticles with glutaraldehyde, and then FDH-ST (SpyTag-fused formate hydrogenase) was immobilized by the spontaneous protein reaction between SpyTag and SpyCatcher to achieve the sequential co-immobilized enzymes FDH-ST-SC- Kg BDH@SiO 2 , which can asymmetrically reduce 2-hydroxyacetophenone to ( R )-1-phenyl-1,2-ethanediol by cofactor regeneration. In comparison to the mixture of free enzymes, the sequential co-immobilized enzymes created under the design conditions performed around 50% activity recovery. Furthermore, FDH-ST-SC- Kg BDH@SiO 2 presented wider pH tolerance and greater organic solvents tolerance than the mixture of free enzymes as well as retained 51.7% of overall activity after being recycled eight times. Finally, the sequential co-immobilized enzyme enabled the biosynthesis of ( R )-1-phenyl-1,2-ethanediol from 80 mM 2-hydroxyacetophenone in the phosphate buffer reaction system, with 87.4% yield and over 99% optical purity.
研究领域:
生物化学
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纳米生物学
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酶工程
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高影响因子
Molecular Catalysis
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2022
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IF 5.089
Xuanru Zhong,Yunjian Ma,Xizhen Zhang,Jiahui Zhang,Bin Wu,Frank Hollmann,Yonghua Wang
Hydrocarbon synthesis from (waste)oils enabled by a cascade of lipase-catalysed hydrolysis and decarboxylase-catalysed decarboxylation has become an active area of research en route to alternative, biobased fuels. However, Poor substrate transport efficiency is a major issue causing low reaction rates. This study focused on a protein self-assembly strategy based on SpyTag/SpyCatcher to overcome diffusion limitations. For this, two fusion proteins, TLL-Linker-SpyCatcher based on the lipase from Thermomyces lanuginosus and Cv FAP-Linker-SpyTag based on the fatty acid photodecarboxylase from Chlorella variabilis were designed. A covalent multi-enzyme complex (TLL- Cv FAP) was formed spontaneously by self-assembly of each enzyme. The effects of temperature, pH and molar ratio of self-assembled components on assembly efficiency were investigated. The results showed that the multi-enzyme complex TLL- Cv FAP reached about 60% after 12 h of assembly, and the enzyme activity of the multienzyme complex was increased by about 50% compared to that of the corresponding non-assembled enzymes. Under optimized conditions 10 mM soybean oil were converted into 25 mM of the corresponding hydrocarbons, suggesting a good potential of biofuel synthesis.
研究领域:
生物技术
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酶工程
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可持续化学
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高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2024
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IF 7.7
Mianxing Luo,Meng Zhang,Changbiao Chi,Guo Chen
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 Ni 2+ , was prepared to explore the self-assembly characteristics of PduQ-SpyTag (P-T) and Nox-SpyCatcher (N C) on its surface, based on the mixed interaction mode consisting the affinity of His-tag/Ni 2+ 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 N C at the interface, a possible multienzyme interaction mechanism for the affinity-assisted covalent self-assembly on the Ni 2+ 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 N C. 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 .
研究领域:
生物化学
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合成生物学
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PubMed
高影响因子
Biotechnology Journal
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2024
·Vol. 19
·期 6
·2400040
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IF 3.2
Shibo Jiang,Ying Lin,Suiping Zheng
IMP (inosinic acid) is a crucial intermediate in the purine metabolic pathway and is continuously synthesized in all cells. Besides its role as a precursor for DNA and RNA, IMP also plays a critical or essential role in cell growth, energy storage, conversion, and metabolism. In our study, we utilized the circularly permuted fluorescent protein (cpFP) and IMP dehydrogenase to screen and develop the IMP biosensor, IMPCP1. By introducing a mutation in the catalytically active site of IMPCP1, from Cys to Ala, we disrupted its ability to catalyze IMP while retaining its capability to bind to IMP without affecting the IMP concentration in the sample. To immobilize IMPCP1, we employed the SpyCatcher/SpyTag system and securely attached it to Magarose-Epoxy, resulting in the development of the IMP rapid test kit, referred to as IMPTK. The biosensor integrated into IMPTK offers enhanced stability, resistance to degradation activity, and specific recognition of IMP. It is also resistant to peroxides and temperature changes. IMPTK serves as a rapid and stable assay for analyzing IMP concentrations in fermentation broth. Within the linear range of IMP concentrations, it can be utilized as a substitute for HPLC. The IMPTK biosensor provides a reliable and efficient alternative for monitoring IMP levels, offering advantages such as speed, stability, and resistance to environmental factors.
研究领域:
生物化学
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生物技术
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分子生物学
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BIOPROCESS AND BIOSYSTEMS ENGINEERING
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2023
·Vol. 46
·期 9
·1365-1373
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IF 3.8
Wang Ru,Jia Jianyao,Liu Xue,Chen Yaru,Xu Qing,Xue Feng
Chiral compounds are important drug intermediates that play a critical role in human life. Herein, we report a facile method to prepare multi-enzyme nano-devices with high catalytic activity and stability. The self-assemble molecular binders SpyCatcher and SpyTag were fused with leucine dehydrogenase and glucose dehydrogenase to produce sc-LeuDH (SpyCatcher-fused leucine dehydrogenase) and GDH-st (SpyTag-fused glucose dehydrogenase), respectively. After assembling, the cross-linked enzymes LeuDH-GDH were formed. The crosslinking enzyme has good pH stability and temperature stability. The coenzyme cycle constant of LeuDH-GDH was always higher than that of free double enzymes. The yield of l - tert -leucine synthesis by LeuDH-GDH was 0.47 times higher than that by free LeuDH and GDH. To further improve the enzyme performance, the cross-linked LeuDH-GDH was immobilized on zeolite imidazolate framework-8 (ZIF-8) via bionic mineralization, forming LeuDH-GDH @ZIF-8. The created co-immobilized enzymes showed even better pH stability and temperature stability than the cross-linked enzymes, and LeuDH-GDH@ZIF-8 retains 70% relative conversion rate in the first four reuses. In addition, the yield of LeuDH-GDH@ZIF-8 was 0.62 times higher than that of LeuDH-GDH, and 1.38 times higher than that of free double enzyme system. This work provides a novel method for developing multi-enzyme nano-device, and the ease of operation of this method is appealing for the construction of other multi-enzymes @MOF systems for the applications in the kinds of complex environment. Graphical abstract
研究领域:
生物化学
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合成生物学
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材料科学
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BIOMEDICAL CHROMATOGRAPHY
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2024
·Vol. 38
·期 9
·e5957
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IF 1.8
Yu Yi,Xuewang Gong,Mengyuan Cui,Yuting Liang,Jianfeng Mei,Guoqing Ying,Yinfei Wu
Microbial metabolites are an important source of tyrosinase (TYR) inhibitors because of their rich chemical diversity. However, because of the complex metabolic environment of microbial products, it is difficult to rapidly locate and identify natural TYR inhibitors. Affinity-based ligand screening is an important method for capturing active ingredients in complex samples, but ligand immobilization is an important factor affecting the screening process. In this paper, TYR was used as ligand, and the SpyTag/SpyCatcher coupling system was used to rapidly construct affinity chromatography vectors for screening TYR inhibitors and separating active components from complex samples. We successfully expressed SpyTag–TYR fusion protein and SpyCatcher protein, and incubated SpyCatcher protein with epoxy-activated agarose. The SpyTag–TYR protein was spontaneously coupled with SpyCatcher to obtain an affinity chromatography filler for immobilization of TYR, and the performance of the packaging material was characterized. Finally, compound 1 with enzyme inhibitory activity was successfully obtained from the fermentation product of marine microorganism C. Through HPLC, MS, 1 H NMR and 13 C NMR analyses, its structure was deduced as azelaic acid, and its activity was analyzed. The results showed that this is a feasible method for screening TYR inhibitors in complex systems.
研究领域:
生物化学
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药物化学
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PubMed
高影响因子
BIOCONJUGATE CHEMISTRY
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2026
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IF 4.5
Liqiang Wei,Hongfei Wang,Chunyue Du,Antony Kam,Shining Loo
We present a one-pot approach for orthogonal dual functionalization of mi3 self-assembling protein nanoparticles by combining SpyCatcher003/SpyTag003 isopeptide bond formation with Sortase A-mediated transpeptidation. The nanoparticles were engineered with N-terminal SpyCatcher003 domains and C-terminal LPETGGH motifs, enabling site-specific conjugation of distinct cargo molecules. To overcome expression limitations, an N-terminal vesicle nucleating peptide (VNP6) was incorporated, which enhanced protein yields while maintaining nanocage integrity. Critical to this strategy, the calcium-independent Sortase A variant (SRT7+) was employed to avoid calcium-induced nanoparticle aggregation, enabling orthogonal ligation under physiological conditions. The platform enabled simultaneous conjugation of HER2-targeting affibodies and fluorescent probes in a single reaction, and these dual-functionalized self-assembling protein nanoparticles exhibited specific cellular uptake in HER2-positive cancer cells. This integrated one-pot approach proved scalable and modular for engineering protein-based nanomaterials for targeted therapy and diagnostics.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2018
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IF 3.412
Jingjing Bao,Nian Liu,Liying Zhu,Qing Xu,He Huang,Ling Jiang
Chitin is used as an essential raw material for the production of glucosamine (GlcN). In this study, we adopted three key enzymes, isolated from Thermococcus kodakaraensis KOD1, that catalyze the sequential conversion of α-chitin into GlcN and developed a multienzyme-assembly-cascade (MAC) system immobilized in a bacterial biofilm, which enabled a multistep one-pot reaction. Specifically, the SpyTag–SpyCatcher and SnoopTag–SnoopCatcher pairs provided covalent and specific binding force to fix enzymes to the biofilm one by one and assemble close enzyme cascades. The MAC system showed great catalytic activity, converting 79.02 ± 3.61% of α-chitin into GlcN with little byproducts, which was 2.09 times that of GlcN catalyzed by a mixture of pure enzymes. The system also exhibited good temperature and pH stability. Notably, 90% of enzyme activity was retained after 6 rounds of reuse, and appreciable activity remained after 17 rounds.
研究领域:
生物化学
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生物技术
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酶学
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PubMed
高影响因子
Biomolecules
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2022
·Vol. 12
·期 7
·997
·
IF 6.064
Mingyu Jia,Zhiyuan Luo,Haomin Chen,Bianqin Ma,Li Qiao,Qinjie Xiao,Pengfei Zhang,Anming Wang
Ideal immobilization with enhanced biocatalyst activity and thermostability enables natural enzymes to serve as a powerful tool to yield synthetically useful chemicals in industry. Such an enzymatic method strategy becomes easier and more convenient with the use of genetic and protein engineering. Here, we developed a covalent programmable polyproteam of tyrosine ammonia lyases (TAL-CLEs) by fusing SpyTag and SpyCatcher peptides into theN-terminal andC-terminal of the TAL, respectively. The resulting circular enzymes were clear after the spontaneous isopeptide bonds formed between the SpyTag and SpyCatcher. Furthermore, the catalytic performance of theTAL-CLEswas measured via a synthesis sample ofp-Coumaric acid. OurTAL-CLEsshowed excellent catalytic efficiency, with 98.31 ± 1.14% yield of the target product—which is 4.15 ± 0.08 times higher than that of traditional glutaraldehyde-mediated enzyme aggregates. They also showed over four times as much enzyme-activity as wild-type TAL does and demonstrated good reusability, and so may become a good candidate for industrial enzymes.Keywords:SpyTag/SpyCatcher;carrier-free immobilized enzyme;crosslinked enzyme protein;cell lysate;tyrosine ammonia lyase;bio-orthogonal system
研究领域:
生物技术
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酶工程
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合成生物学
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PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
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2021
·
IF 3.978
Mianxing Luo,Meng Zhang,Guo Chen,Jun Zhao,Hongwei Guo
Functional carrier capable of covalently binding proteins from crude lysate under mild condition has become increasingly important in bioprocess. In this paper, a magnetic microbead that can covalently immobilize specific proteins directly from crude protein solution was developed, and its application conditions and stability were evaluated. The SpyCatcher was firstly modified with N-terminal cysteine by genetic methods and then the cys-SpyCatcher was covalently immobilized on epoxy-modified magnetic beads by epoxy-thiol reaction. The reaction between the epoxy and the only terminal thiol group in cys-SpyCatcher ensured the same orientation of the cys-SpyCatcher on the particle surface, while the maximum binding capacity of cys-SpyCatcher reached 23.6 μg/mg under the condition of 250 mM Na 2 SO 4 , pH 8.5 at initial cys-SpyCatcher concentration of 3 mg/mL. The functional magnetic microsphere ( [email protected] ) showed covalent site-specific capture property for enhanced green fluorescent protein fused with SpyTag (SpyTag-EGFP). A maximum binding capacity of (25 μg SpyTag-EGFP)/(mg beads) was far more than that of the previous carrier with SpyCatcher. In addition, the [email protected] could specifically capture the SpyTagged protein directly from the cell lysate, and this carrier showed good stability at pH 4–9 and temperature of 30–70ºC. Therefore, as a universal covalent capture carrier, it is believed that the [email protected] is useful in one-step purification and site-specific immobilization of the SpyTagged protein in bioprocess or biomedical application.
研究领域:
生物技术
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生物化学
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蛋白质工程
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高影响因子
Foods
·
2025
·Vol. 14
·期 24
·
IF 5.1
Tailong Wei,Xiao Chen,Chong Cai,Yuanzhen Guo,Mengjun Zhou,Qiannan Gao,Qinghua He
Mimotope-based immunoassays offer an eco-friendly alternative to chemically synthesized antigens for the quantitative analysis of small molecules, but their use for practical on-site and high-throughput residue monitoring remains limited. Herein, we report the selection, production, and application of a phage display–derived mimotope targeting an anti-uniconazole monoclonal antibody (UCZ-mAb), with the aim of developing two complementary immunoassays that enable sensitive, eco-friendly detection of UCZ residues in agricultural samples. A 12-mer phage-displayed peptide library was screened to identify UCZ-specific mimotopes, and a selected sequence was genetically fused to SpyTag and expressed inEscherichia colito generate a SpyTagged mimotope. Leveraging the SpyCatcher/SpyTag self-assembly system, the SpyTagged mimotope was directionally conjugated onto SpyCatcher-functionalized time-resolved fluorescence beads (TRFBs) and subsequently used as a signal-labeled competitive antigen in a lateral flow immunoassay (LFIA) designed for rapid on-site screening. In parallel, a wash-free magnetic separation immunoassay (MSIA) suitable for green, high-throughput screening in routine laboratories was established using self-assembled mimotope-TRFB probes. The LFIA and MSIA exhibited half-maximal inhibitory concentrations (IC50) of 3.70–6.72 μg/kg and 16.4–18.3 μg/kg, respectively, in real samples. Spiked-sample recoveries ranged from 91.1 to 107.8% for LFIA and 92.6–115.7% for MSIA, demonstrating acceptable accuracy and precision. These results indicate that the SpyTagged mimotope–based LFIA and MSIA provide complementary, reliable, and sensitive platforms for on-site screening and high-throughput monitoring of UCZ residues in agricultural samples, while avoiding the drawbacks associated with traditional chemical antigen synthesis.
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