PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
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2018
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IF 1.241
Mingcong Shao,Lili Xiu,Haijiang Zhang,Jianying Huang,Xingwen Gong
Chitosan/cellulose-based beads (CCBs) for the affinity purification of histidine-tagged proteins were prepared from chitosan/cellulose dissolved in ionic liquid as a solvent, and their structures were characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and thermogravimetric analysis. The affinity purification was used to separate hexahistidine-tagged (his-tagged) enhanced green fluorescent protein (EGFP) from Escherichia coli. The results showed that Zn2+–CCB exhibited more specific adsorption capacity toward the target protein compared with Ni2+–CCB and Cu2+–CCB. The maximum adsorption of EGFP was 1.84 mg/g of Zn2+–CCB, with 90% purity under the optimized conditions (ionic strength (1.0 M NaCl), pH (7.2) and imidazole concentration (500 mM)). In addition, a regeneration method for the sorbent was further developed by washing with ethylenediaminetetraacetic acid disodium and then reimmobilizing with metal ions. This technique is an alternative method for the purification of his-tagged proteins, making the process more economical, fast, stable, and large batch.
研究领域:
生物化学
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蛋白质工程
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DOI
PubMed
高影响因子
PROCESS BIOCHEMISTRY
·
2019
·
IF 2.883
Fuhao Yang,Junfen Wan,Xuejun Cao
Thermo-responsive polymers have great potential for industrial applications of bio-separation and purification. However, the poor affinity and low recovery of thermoresponsive copolymers are the main factors limiting their large-scale application. In this paper, a recyclable thermoresponsive affinity copolymer (P NHM -IDA-Ni 2+ ) was prepared by immobilizing nickel ions (Ni 2+ ) on copolymer P NHM , and P NHM -IDA-Ni 2+ was applied to the purification of ε-polylysine. The lower critical solution temperature (LCST) of P NHM and P NHM -IDA-Ni 2+ were 31.0 °C and 34.0 °C, respectively. Additionally, the recoveries of both copolymers were over 95.0%. The main parameters, such as pH were investigated to optimize the adsorption conditions. In addition, the Langmuir and Freundlich adsorption models were used to predict the maximum adsorption capacity of ε-polylysine. The results of the affinity precipitation demonstrated that the maximum adsorption capacity was 42.9 mg/g, and the adsorption was considered to follow the mono-layer model. The thermodynamic parameters (ΔG 0 , ΔH 0 and ΔS 0 ) of the ε-polylysine adsorption indicated that the adsorption was spontaneous and exothermic. The maximum elution recovery (93.5%) was achieved at pH 5.0 with 0.2 M imidazole. The results of tricine-SDS-PAGE and HPLC (Purity: increased from 84% to 99%) showed that the ε-polylysine was well separated from the crude extract by using the affinity copolymer P NHM -IDA-Ni 2+ .
研究领域:
生物工程
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分离纯化技术
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高分子化学
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产品关联
DOI
高影响因子
ANALYTICAL CHEMISTRY
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2020
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IF 6.785
Guizhen Liu,Tao Fu,Ying Han,Shichen Hu,Xuepei Zhang,Mengmeng Zheng,Piliang Hao,Lifeng Pan,Jingwu Kang
We describe an affinity purification–mass spectrometry (AP–MS) method for probing the interactome of a special targeting protein. The AP was implemented with monolithic micro immobilized metal ion affinity chromatography columns (m-IMAC) which were prepared by photoinitiated polymerization in the tip of a pipet (spin-tip columns). The recombinant His6-tagged protein (bait protein) was reversibly immobilized on the affinity column through the chelating group nitrilotriacetic acid (NTA)–Ni2+. The bait protein and its interacting partners can be easily eluted from the affinity matrix. The pulled-down cellular proteins were then analyzed with label-free quantitative proteomics. We used this method for probing the interactome concerning the GOLD (Golgi dynamics) domain of the autophagy-associated adaptor protein FYCO1. Totally, 96 proteins including seven literature-reported FYCO1-associating proteins were identified. Among them CCZ1 and MON1A were further biochemically validated, and the direct interaction between the FYCO1 GOLD domain with CCZ1 was confirmed by co-immunoprecipitation experiments.
研究领域:
蛋白质组学
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生物化学
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细胞生物学
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产品关联
DOI
PubMed
高影响因子
STAR Protocols
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2022
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IF 0
Weixiang Bian,Hua Jiang,Shan Feng,Junjie Chen,Wenqi Wang,Xu Li
Summary Identification of protein interactors is fundamental to understanding their functions. Here, we describe a modified protocol for tandem affinity purification coupled with mass spectrometry (TAP/MS), which includes two-step purification. We detail the S-, 2×FLAG-, and Streptavidin-Binding Peptide (SBP)- tandem tags (SFB-tag) system for protein purification. This protocol can be used to identify protein interactors and establish a high-confidence protein–protein interaction network based on computational models. This is particularly useful for identifying bona fide interacting proteins for subsequent functional studies. For complete details on the use and execution of this protocol, please refer to Bian et al. (2021) .
研究领域:
分子生物学
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蛋白质组学
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细胞生物学
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DOI
PubMed
NEW JOURNAL OF CHEMISTRY
·
2021
·Vol. 45
·期 15
·6817-6825
·
IF 3.591
Jiawei Liu,Yingying Liu,Yixun Liang,Fen Ma,Quan Bai
In this work, we prepared poly-L-lysine (PLL) functionalized magnetic graphene through a simple covalent bonding process. The composite was further conjugated with aspartic acid (Asp) and chelated copper ions to obtain a metal affinity PLL-functionalized magnetic graphene composite (MGPLA-Cu). Various characterizations via FT-IR, XRD, TGA, VSM and SEM/TEM confirm its physicochemical properties. Owing to the strong metal affinity forces between metal affinity groups and the residual histidine in proteins, the MGPLA-Cu composite exhibits highly selective adsorption performance toward histidine-rich proteins, i.e., hemoglobin (Hb) in this case. A high adsorption efficiency of ca. 100% was achieved for 200 mg L−1 Hb in a phosphate buffer at pH 8. The equilibrium adsorption isotherm of Hb on the MGPLA-Cu composite well fitted to the Langmuir theoretical model (R2 = 0.9969), along with the maximum adsorption capacity (qmax) at 334 mg g−1. The adsorption kinetics was favorably described by the pseudo-second-order kinetic model. The retained Hb could be effectively recovered with a 0.2 mol L−1 carbonate buffer containing 0.05 mol L−1 imidazole at pH 10, giving rise to a recovery of ca. 97%. The MGPLA-Cu composite showed favorable reusability, and a high adsorption efficiency (>80%) was maintained after five adsorption–desorption operation cycles. The practical application demonstrates that the MGPLA-Cu composite can isolate Hb from human whole blood with high selectivity and high purity.
研究领域:
材料科学
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生物化学
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蛋白质工程
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产品关联
DOI
高影响因子
JOURNAL OF CHROMATOGRAPHY A
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2015
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IF 4.169
Hu Liu,Xiaoyan Dong,Yan Sun
A series of highly charged nanoscale chelators were fabricated by grafting of poly(glycidyl methacrylate-iminodiacetic acid) (pGI) chains with iminodiacetic acid (IDA) chelating group on silica nanoparticles (SNPs) via atom transfer radical polymerization (ATRP). The nanoscale chelators, denoted as SNPs-pGI, possessed a nickel ion chelating capacity as high as 2800 μmol/g, 50 times higher than the IDA-modified Sepharose FF (IDA-Sepharose) resin reported in literature and offered a high affinity binding capacity for hexahistidine-tagged enhanced green fluorescence protein (6 × His-EGFP) after nickel ion loading. More importantly, the anionic SNPs-pGI of high charge densities displayed much better performance than IDA-Sepharose in facilitating the refolding of like-charged 6 × His-EGFP from inclusion bodies (IBs). For example, for 0.2 mg/mL 6 × His-EGFP IB refolding, addition of 6.2 μL/mL SNPs-pGI with the highest charge density led to a refolding yield of 90%, over 43% higher than that obtained with 460 μL/mL IDA-Sepharose. It is notable that the much higher efficiency of the nanoscale chelator was obtained with a chelator consumption corresponding to only 1.4% of IDA-Sepharose. Moreover, the highly charged SNPs-pGI could efficiently facilitate the refolding of 6 × His-EGFP at higher IB concentrations (0.4 and 0.8 mg/mL). After refolding, nickel ions addition led to the recovery of the refolded 6 × His-EGFP with high yield (80%), purity (96%) and enrichment ratio (1.8). All the results suggest that the SNPs-pGI of high charge densities were promising for cost-effective recovery of His-tagged proteins expressed as IBs with the integrative like-charge facilitated refolding and metal-chelate affinity purification strategy.
研究领域:
生物化学
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纳米技术
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蛋白质工程
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DOI
PubMed
高影响因子
Journal of Advanced Research
·
2024
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IF 11.4
Ya-Bo Zhao,Shi-Zhe Wang,Wen-Ting Guo,Le Wang,Xun Tang,Jin-Nan Li,Lin Xu,Qi-Xin Zhou
Introduction Subtypes of the dipeptidyl peptidase (DPP) family, such as DPP4, are reportedly associated with memory impairment. DPP9 is widely distributed in cells throughout the body, including the brain. However, whether DPP9 regulates memory has not yet been elucidated. Objectives This study aimed to elucidate the role of DPP9 in memory, as well as the underlying molecular mechanism. Methods We performed immunofluorescence on mouse brains to explore the distribution of DPP9 in different brain regions and used AAV vectors to construct knockdown and overexpression models. The effects of changing DPP9 expression on memory were demonstrated through behavioral experiments. Finally, we used electrophysiology, proteomics and affinity purification mass spectrometry (AP-MS) to study the molecular mechanism by which DPP9 affects memory. Results Here, we report that DPP9, which is found almost exclusively in neurons, is expressed and has enzyme activity in many brain regions, especially in the hippocampus. Hippocampal DPP9 expression increases after fear memory formation. Fear memory was impaired by DPP9 knockdown and enhanced by DPP9 protein overexpression in the hippocampus. According to subsequent hippocampal proteomics, multiple pathways, including the peptidase pathway, which can be bidirectionally regulated by DPP9. DPP9 directly interacts with its enzymatic substrate neuropeptide Y (NPY) in neurons. Hippocampal long-term potentiation (LTP) is also bidirectionally regulated by DPP9. Moreover, inhibiting DPP enzyme activity impaired both LTP and memory. In addition, AP-MS revealed that DPP9-interacting proteins are involved in the functions of dendritic spines and axons. By combining AP-MS and proteomics, DPP9 was shown to play a role in regulating actin functions. Conclusion Taken together, our findings reveal that DPP9 affects the CNS not only through enzymatic activity but also through protein–protein interactions. This study provides new insights into the molecular mechanisms of memory and DPP family functions.
研究领域:
神经科学
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分子生物学
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行为科学
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蛋白质组学
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DOI
PubMed
高影响因子
Microbiology Spectrum
·
2022
·Vol. 10
·期 3
·
IF 9.043
Meng Wang,Yujiao Tian,Li Xu,Fusheng Zhang,Huigai Lu,Moran Li,Bei Li
Klebsiella pneumoniae is a pathogen known for its high frequency of antimicrobial resistance. Responses to various environmental stresses during its life can influence the resistance to antibiotics. Here, we demonstrate the role and mechanism of KbvR regulator in the response to environmental osmotic stress and in the effect of osmotic stress on antimicrobial resistance. The kbvR mutant strain exhibited increasing tolerance to high osmotic stress and certain antibiotics, including β-lactams. The expression levels of KbvR and outer membrane porin OmpK36 were upregulated in response to high osmotic stress in the wild type (WT), and the deletion of kbvR decreased the expression level of ompK36. The membrane permeability of the kbvR mutant strain was decreased, which was partly restored through the upregulated expression of OmpK36. The DNA affinity purification sequencing (DAP-seq) and microscale thermophoresis (MST) assay disclosed the binding of KbvR to the promoter of the ompK36 gene, indicating that KbvR directly and positively regulated the expression of OmpK36. The high osmotic stress increased the susceptibility to β-lactams and the expression of ompK36 in the WT strain. However, the increased ompK36 expression and the susceptibility to β-lactams in the kbvR mutant strain under high osmotic stress were lower than those of WT. In conclusion, our study has identified that high osmotic stress in the environment influenced the resistance of K. pneumoniae to antibiotics and that the regulation of KbvR with OmpR on the expression of OmpK36 was involved in countering high osmotic stress to change the antimicrobial resistance.IMPORTANCE Klebsiella pneumoniae is considered a global threat because of the rising prevalence of multidrug-resistant strains and their optimal adaptation to clinical environments and the human host. The sensing and adaption abilities of bacteria to the environmental osmotic stress can change the expression of their outer membrane porins, membrane permeability, and resistance to antibiotics. This study reports that KbvR is a newly found regulator that can be upregulated under high osmotic stress and directly regulate the expression of OmpK36 to change the resistance of K. pneumoniae to β-lactam antibiotics. The results demonstrate how adaptation to high osmotic stress changes the sensitivity of K. pneumoniae to antibiotics. The mechanism can be used to sensitize bacteria to antibiotics and highlight new potential strategies for exploiting shared constraints in governing adaptation to diverse environmental challenges.
研究领域:
微生物学
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分子生物学
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细菌遗传学
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抗菌素耐药性
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DOI
高影响因子
Food Bioscience
·
2025
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IF 5.9
Yuling Tang,Xunjiao Wei,Yonghua Wang,Wen Chen
Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are principal omega-3 polyunsaturated fatty acids (PUFAs) nutritional and pharmaceutical interest. However, their near-isomeric structures render large-scale separation technically demanding and costly. Here we evaluate human fatty-acid-binding protein 7 (FABP7) as a biorecognition element capable of discriminating between these two lipids. Recombinant FABP7, expressed and purified from Escherichia coli , was analyzed for its binding affinity toward DHA and EPA by bio-layer interferometry (BLI). FABP7 bound DHA with an equilibrium dissociation constant K d = 80.18 ± 3.71 mmol/m 3 , whereas binding to EPA was 7.5-fold weaker (K d = 600.2 ± 30.2 mmol/m 3 ). Competitive BLI experiments confirmed that DHA efficiently displaced pre-bound EPA, while EPA was largely unable to remove DHA even at millimolar excess. These findings demonstrate that FABP7 possesses intrinsic molecular selectivity for DHA over EPA and could therefore serve as the affinity component of bio-based fractionation platforms. This work establishes a quantitative framework for engineering FABP-derived resins or sensors aimed at sustainable, low-energy purification of high-value ω-3 lipids.
研究领域:
分子生物学
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脂质代谢
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生物化学
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结构生物学
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生物分离工程
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产品关联
DOI
高影响因子
JOURNAL OF COLLOID AND INTERFACE SCIENCE
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2015
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IF 3.368
Ling Yu,Zhuan Zhuan Shi,Chang Ming Li
Poly[glycidyl methacrylate- co -poly (ethylene glycol) methacrylate] microspheres for the first time were successfully synthesized by atom transfer radical polymerization (ATRP) method at room temperature. The co-polymerization approach was investigated to delicately control the microsphere morphology and size-distribution by reaction conditions including solvent percentage, monomer loading and rotation speed. The results show that the average size of the microspheres is ∼5.7 μm with coexistence of epoxy, hydroxyl and ether groups, which provide plentiful functional sites for protein anchoring. The mechanism of the microsphere formation is proposed. The microsphere successfully demonstrates its unique application for affinity purification of proteins, in which the functional epoxy group facilitates a simple and efficient protein covalent immobilization to purify immunoglobulin G on the microspheres, while the hydrophilic poly (ethylene glycol) motif can repulse nonspecific protein adsorption for good specificity. This microspheres can be used in broad protein biosensors due to their abundant functional groups and high surface to volume ratio.
研究领域:
材料科学
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化学工程
·
生物化学
·
生物技术
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产品关联
DOI
PubMed
高影响因子
Nature Communications
·
2024
·Vol. 15
·期 1
·1-18
·
IF 14.7
Shao Xinyang,Tian Meng,Yin Junlong,Duan Haifeng,Tian Ye,Wang Hui,Xia Changsheng,Wang Ziwei,Zhu Yanxi,Wang Yifan,Chaihu Lingxiao,Tan Minjie,Wang Hongwei,Huang Yanyi,Wang Jianbin,Wang Guanbo
The characterization of protein complex is vital for unraveling biological mechanisms in various life processes. Despite advancements in biophysical tools, the capture of non-covalent complexes and deciphering of their biochemical composition continue to present challenges for low-input samples. Here we introduce SNAP-MS, a Stationary-phase-dissolvable Native Affinity Purification and Mass Spectrometric characterization strategy. It allows for highly efficient purification and characterization from inputs at the pico-mole level. SNAP-MS replaces traditional elution with matrix dissolving during the recovery of captured targets, enabling the use of high-affinity bait-target pairs and eliminates interstitial voids. The purified intact protein complexes are compatible with native MS, which provides structural information including stoichiometry, topology, and distribution of proteoforms, size variants and interaction states. An algorithm utilizes the bait as a charge remover and mass corrector significantly enhances the accuracy of analyzing heterogeneously glycosylated complexes. With a sample-to-data time as brief as 2 hours, SNAP-MS demonstrates considerable versatility in characterizing native complexes from biological samples, including blood samples.
研究领域:
生物物理学
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蛋白质组学
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分析化学
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分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Foods
·
2022
·Vol. 11
·期 8
·1155
·
IF 5.561
Jie-Biao Guo,Jin-Sheng Cheng,Tai-Long Wei,Fan-Min Wu,Gui-Hong Tang,Qing-Hua He
An immuno-separated assay for ochratoxin A detection coupled with a nano-affinity cleaning up for LC-confirmation was developed. Firstly, ochratoxin A was modified to quantum dot beads for immuno-fluorescent reporters. Secondly, Fe3O4magnetic nanoparticles were conjugated with protein G for immuno-magnetic adsorbents. The immuno-separation of fluorescent reporters by magnetic adsorbents could be completed by ochratoxin A, so the fluorescent reporters released from the immune complex indicate a linear correlation with the concentration of ochratoxin A. Furthermore, the immuno-separated ochratoxin A can be eluted from magnetic adsorbent for LC-conformation. The optimized assay showed results as follows: the quantitative range of the immuno-separated assay was 0.03–100 ng mL−1of ochratoxin A. The recoveries for spiked samples ranged from 78.2% to 91.4%, with the relative standard deviation (RSD) being 11.9%~15.3%. Statistical analysis indicated no significant difference between the HPLC-FLD results based on commercial affinity column and by nano-affinity cleaning up.Keywords:immuno-separated assay;nano-affinity cleaning up;immuno-magnetic adsorbent;immuno-fluorescent reporters;LC-confirmation
研究领域:
分析化学
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毒理学
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食品科学
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）