Bio-protocol
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2025
·Vol. 15
·期 8
·e5270
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IF 1.1
Xiaofeng Yang,Zhanglin Lin,Ya Xiang,Binrui Chen,Zisha Lao
Protein purification is a critical step in both life sciences and biomanufacturing. Traditional affinity chromatography (AC) methods, including His-tag-based purification, provide high-purity proteins but are limited by the high cost of resins and the need for additional tag-removal steps. In this protocol, we present a reusable SpyDock-modified epoxy resin coupled with a pH-inducible self-cleaving intein for direct purification of proteins with authentic N-termini. This method enables efficient protein purification from cell lysates, achieving high purity (>90%) and yields comparable to the His-tag approach, without requiring tag removal. The SpyDock-modified resin protocol is robust, easy to implement, and cost-effective, making it suitable for both research and large-scale industrial applications. Key features • This protocol offers a robust and straightforward method for purifying proteins with authentic N-termini, eliminating the need for additional tag removal steps. • The approach achieves higher purity and comparable yields to the commercial His-tag method. • The SpyDock-modified epoxy resin is easy to prepare, cost-effective, and reusable.
研究领域:
生物化学
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分子生物学
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PubMed
高影响因子
ANALYTICA CHIMICA ACTA
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2020
·
IF 5.977
Zhouxian Li,Qi Wang,Jiawei Mao,Lingyi Zhang,Weibing Zhang,Mingliang Ye
A challenge for shotgun proteomics is the identification of low abundance proteins, which is always hampered owing to the extreme complexity of protein digests and highly dynamic concentration range of proteins. To reduce the complexity of the peptide mixture, we developed a novel method to selectively enrich N-terminal proline peptides via hydrazide chemistry . This method consisted of ortho -phthalaldehyde (OPA) blocking of primary amines in peptides, reductive glutaraldehydation of N-terminal proline and solid phase hydrazide chemistry enrichment of aldehyde-modified N-terminal proline peptide. After enrichment, the number of detected peptides containing N-terminal proline increased from 1304 to 4039 and the ratio of N-terminal proline peptides jumped from 4.4% to 93.7%, showing good enrichment specificity towards N-terminal proline peptides. Besides, the ratio of identified peptides to proteins was decreased from 7.8 (29751/3811) to 1.5 (4347/2821), indicating that sample complexity was drastically reduced through this method. As a result, this novel approach for enriching N-terminal proline peptides is effective in identification of low abundance protein owing to the reduction of sample complexity.
研究领域:
生物化学
·
蛋白质组学
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PubMed
高影响因子
Journal of Translational Medicine
·
2025
·Vol. 23
·期 1
·1-18
·
IF 7.5
Tan Yanzhen,Li Hong,Cao Guojie,Xin Jialin,Yan Dongxu,Liu Yingying,Li Panpan,Zhang Yuxi,Shi Lei,Zhang Bing,Yi Wei,Sun Yang
Background In developed nations, myocardial infarction (MI) is one of the main causes of morbidity and mortality, resulting in a significant economic burden and becoming a global public health problem. C1q/tumor necrosis factor-related protein 9 (CTRP9) is a secreted protein comprising a variable domain, a collagenous region, and a C-terminal trimerizing globular C1q (gC1q) domain. In vivo, the full-length CTRP9 (fCTRP9) can be cleaved into the globular domain of CTRP9 (gCTRP9). Here, we tested the cardio-protective impacts of fCTRP9, gCTRP9, and N-terminal domain, including the variable and collagenous domain, of CTRP9 (nCTRP9) in the context of MI.Methods Studies comparing the protective properties of fCTRP9 and gCTRP9 against MI in mice hearts were performed both in vitro and in vivo. The role of matrix metalloproteinase-9 (MMP9) in CTRP9 cleavage was examined, and the effects of different CTRP9 domains on cardiac fibrosis and cardiac fibroblast (CF) activation were investigated.Results gCTRP9 exerted better protective effects than fCTRP9 against MI, demonstrating superior anti-apoptotic and anti-fibrotic properties. fCTRP9 was cleaved by MMP9, resulting in gCTRP9 and nCTRP9. MMP9 overexpression enhanced the cardioprotective effects of fCTRP9, while nCTRP9 supplementation aggravated cardiac fibrosis in MI mice. Mechanistically, nCTRP9 activated CFs via an increase in Rap1 expression and MEK 1/2 and ERK1/2 phosphorylation.Conclusion sDifferent domains of CTRP9 have distinct cardioprotective effects. gCTRP9 shows beneficial effects, while nCTRP9 promotes cardiac fibrosis. These findings highlight the importance of CTRP9 in cardiac function regulation and suggest prospective therapeutic options for MI treatment.
研究领域:
心脏病学
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分子生物学
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药理学
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PubMed
高影响因子
BIOORGANIC CHEMISTRY
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2025
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IF 4.7
Shuaishuai Cui,Cunxin Guo,Liang Yan,Yujian He,Li Wu
The incorporation of D-amino acids can influence the structure and enzymatic stability of proteins or peptides, especially when they are introduced at enzyme cleavage sites, significantly inhibiting the enzymatic hydrolysis of peptides. The abnormal accumulation of these peptides has been linked to age-related conditions, including cataracts and Alzheimer's disease. N-terminal acetylation, an essential post-translational modification, significantly enhances the physicochemical properties of peptides and plays an essential role in regulating their performance within biological systems. This research examined the impact of N-terminal acetylation on the enzymatic hydrolysis of peptides incorporating D-amino acids. Enzymatic activity assessments showed that N-terminal acetylation greatly promoted the enzymatic breakdown of these peptides by Proteinase K (PROK), with the substrate decay rate constant for the acetylated peptide Ac-6-w increasing by 17.5 times. This enhancement was specific to serine-type proteases, which exhibited a comparable cleavage pattern. Molecular docking further demonstrated that N-terminal acetylation improved interactions within the catalytic triad of serine proteases, leading to faster enzymatic degradation. The results provide novel insights into the impact of N-terminal acetylation on the enzymatic behavior of peptides incorporating D-amino acids, and they propose a potential approach for targeting these peptides to preserve normal physiological functions.
研究领域:
生物化学
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医学
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分子生物学
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PubMed
高影响因子
Science Advances
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2025
·Vol. 11
·期 32
·
IF 12.5
Runchuan Yan,Yan Zhang,Jingjing Zhang,Xiangyi Zhang,Yue Han,Mengfei Wang,Dan Wang,Shengnan Huang,Wei Liu,Qi Shi,Xiaoping Dong,Wen-Quan Zou,Zhen Li,Jiyan Ma
Rapid neurodegeneration distinguishes prion disease from other neurodegenerative disorders. Notably, normal prion protein (PrP C ) is essential for prion-induced rapid neurodegeneration, but the underlying mechanism remains unknown. Here, we show that the unstructured N-terminal region of PrP C induces rapid and lethal neurodegeneration in mice, accompanied by the hallmark of prion disease, spongiosis. The neurotoxic N-terminal PrP is soluble, associates peripherally with lipid membranes, and induces neurotoxicity only when a critical threshold is exceeded. Both the N-terminally localized KKRPKP sequence and octarepeats contribute to neurotoxicity, with KKRPKP being essential. Without it, the N-terminal PrP is innocuous but exacerbates either neurodegeneration caused by N-terminal PrP or neurodegeneration in prion disease induced by intracerebral prion inoculation in mice. Our findings establish that soluble N-terminal PrP causes rapid neurodegeneration in prion disease and is a target for intervention.
研究领域:
神经生物学
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分子生物学
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病理学
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转化医学
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蛋白质错误折叠疾病
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高影响因子
CHEMICAL PAPERS
·
2022
·Vol. 76
·期 6
·3937-3947
·
IF 2.146
Li Xinxin,Lin Min,Zhang Hongfeng,Ji Wenquan,Shi Yuhan,Qi Zhiyang,Fu Tianwen,Li Qinran,Deng Qiliang
A kind of surface imprinted particles was synthesized to recognize cytochrome c (Cyt c) by a novel strategy combining reversible addition-fragmentation chain transfer (RAFT) radical polymerization and epitope surface imprinting. N-terminal epitope nonapeptide of Cyt c was chosen as the template. The selectivity of these particles for protein recognition was obviously improved due to the selection of epitopes rather than the whole protein as templates and the controllable length of polymer shell chain. After epitope had been modified on the surface of silica, the surface imprinted polymer with controlled imprinted layer was synthesized with monomers and cross-linkers by RAFT radical polymerization strategy. The epitope surface imprinted particles were obtained after the peptide removed and trithioester group destruction by hexylamine. The binding capacity of N-terminal peptide reached 1.88 mg peptide per gram with imprinting factor (IF) as 2.42 while the binding capacity of Cyt c reached 8.89 mg protein per gram with IF as 1.71, which were obviously higher than the IF of the material without RAFT strategy. Moreover, Cyt c could be selectively recognized by the epitope surface imprinted particles even in presence of the competitive proteins with different molecular weight and isoelectric point. The performance of protein recognition remained 90% after five cycles of adsorption and desorption. All these results demonstrated that the epitope surface imprinted particles prepared by RAFT strategy are promising to achieve the protein recognition with higher recognition ability, selectivity and reusability.
研究领域:
生物化学
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分子生物学
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高影响因子
Animal Models and Experimental Medicine
·
2024
·
IF 3.8
Xue Wang,Chunyou Ning,Xingyi Cheng,Zhengzhong Wu,Dongbo Wu,Xuemei Ding,Cunxiang Ju,Zhihang Zhou,Lingfeng Wan,Wei Zhao,Peiliang Shi
Background The emerging incidence of pathogenic liver conditions is turning into a major concern for global health. Induction of pyroptosis in hepatocytes instigates cellular disintegration, which in turn liberates substantial quantities of pro-inflammatory intracellular substances, thereby accelerating the advancement of liver fibrosis. Consequently, directing therapeutic efforts towards inhibiting pyroptosis could potentially serve as an innovative approach in managing inflammation related chronic hepatic disorders. Methods GSDMD-NT ki/wt mice and Alb-cre ki/wt mice were generated using CRISPR/Cas9 technology. After crossing the two strains together, we induced conditional cell death by doxycycline to construct a mouse model of liver fibrosis. We analyzed differentially expressed genes by RNA sequencing and explored their biological functions. The efficacy of obeticholic acid (OCA) in the treatment of liver fibrosis was assessed. Results Doxycycline-treated GSDMD-NT ki/wt × Alb-cre ki/wt mice showed severe liver damage, vacuolation of hepatocytes, increased collagen fibers, and accumulation of lipid droplets. The expression of liver fibrosis related genes was greatly increased in the doxycycline-treated mouse liver compared with untreated mouse liver. RNA-sequencing showed that upregulated differentially expressed genes were involved in inflammatory responses, cell activation, and metabolic processes. Treatment with OCA alleviated the liver fibrosis, with reduced ALT and AST levels seen in the GSDMD-NT ki/wt × Alb-cre ki/wt mice. Conclusions We successfully constructed a novel mouse model for liver fibrosis. This GSDMD-NT-induced fibrosis may be mediated by abnormal lipid metabolism. Our results demonstrated that we successfully constructed a mouse model of liver fibrosis, and GSDMD-NT induced fibrosis by mediating lipid metabolism.
研究领域:
肝病学
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分子生物学
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遗传学
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PubMed
高影响因子
PROTEIN EXPRESSION AND PURIFICATION
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2022
·
IF 2.025
Yi Rong,Chaofeng Zhang,Wen-Chao Gao,Cheng Zhao
The main protease (M pro ) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a vital role in viral replication. To study the function of M pro and screen inhibitors targeting M pro , it is necessary to prepare high-purity and high-activity M pro . In this study, four types of SARS-CoV-2 M pro s containing different termini were prepared, and their activities were determined successfully. The results showed that the activity of wild-type (WT) M pro was the highest, and the additional residues at the N-terminus but not at the C-terminus had a major effect on the enzyme activity. To explain this, the alignment of structures of different forms of M pro was determined, and the additional residues at the N-terminus were found to interfere with the formation of the substrate binding pocket. This study confirms the importance of the natural N-terminus to the activity of M pro and suggests that WT-GPH 6 (M pro with eight additional residues at the C-terminus) can be used as a substitute for authentic M pro to screen inhibitors. In short, this study provides a reference for the expression and purification of new coronaviruses confronted in the future.
研究领域:
病毒学
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生物化学
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分子生物学
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PubMed
高影响因子
FOOD CONTROL
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2020
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IF 4.258
Xuejian Yu,Naiyan Lu,Jiyue Wang,Zhe Chen,Chen Chen,Joe Mac Regenstein,Peng Zhou
Nisin, the only antimicrobial peptide that has GRAS (generally considered as safe) status from both WHO and FDA, has the potential to become an important natural antibiotic as bacteria become more resistant to traditional antibiotics. Many studies have focused on how modification of nisin affects antimicrobial activity. In this study, nisin and chitosan were crosslinked using genipin to modify the N-terminal of nisin. The antimicrobial experiments were carried out on Staphylococcus aureus ATCC 6538. The results showed that nisin still retained antimicrobial activity after modification. When the crosslinking ratio of nisin to chitosan increased to 200: 1, MIC decreased from 48 μg/ml to 40 μg/ml. Less chitosan increased local number density and antimicrobial activity of nisin. When the local number density was high, adjacent peptides could cooperatively penetrate into the lipid membrane. The results of this study may help to better understand the antimicrobial mechanism and provide suggestions for the further modification of nisin.
研究领域:
微生物学
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药学
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生物技术
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高影响因子
MOLECULAR CELL
·
2022
·
IF 19.328
Di Chen,Mohan Lyu,Xiaoxia Kou,Jing Li,Zhixuan Yang,Lulu Gao,Yue Li,Liu-min Fan,Hui Shi,Shangwei Zhong
Summary Light and temperature in plants are perceived by a common receptor, phytochrome B (phyB). How phyB distinguishes these signals remains elusive. Here, we report that phyB spontaneously undergoes phase separation to assemble liquid-like droplets. This capacity is driven by its C terminus through self-association, whereas the intrinsically disordered N-terminal extension (NTE) functions as a biophysical modulator of phase separation. Light exposure triggers a conformational change to subsequently alter phyB condensate assembly, while temperature sensation is directly mediated by the NTE to modulate the phase behavior of phyB droplets. Multiple signaling components are selectively incorporated into phyB droplets to form concentrated microreactors, allowing switch-like control of phyB signaling activity through phase transitions. Therefore, light and temperature cues are separately read out by phyB via allosteric changes and spontaneous phase separation, respectively. We provide a conceptual framework showing how the distinct but highly correlated physical signals are interpreted and sorted by one receptor.
研究领域:
植物生物学
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分子生物学
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细胞生物学
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PubMed
高影响因子
Insects
·
2024
·Vol. 15
·期 7
·501
·
IF 2.7
Ningna Yin,Dan Shen,Yinlan Liang,Pengfei Wang,Yonghe Li,Naiyong Liu
Simple SummaryThe swallowtail butterflyPapilio xuthusis a specialized herbivorous insect, with its larvae solely utilizing the Rutaceae plants as hosts. The host seeking and orientation of this butterfly mainly rely on chemosensory-related genes expressed in the antennae, including chemosensory proteins (CSPs). Here, we characterized the putative roles of a female-antenna-biased PxutCSP19 inP. xuthusassociated with the perception of host volatiles and the sequestering of insecticides. This study examined the key functions of PxutCSP19 in the interactions of six host-derived odorants and eight insecticides. Notably, an extended N-terminus of PxutCSP19 did not significantly affect the binding specificities of this protein to ligands with high affinities. Our findings provide insight into the binding mechanisms of PxutCSP19 inP. xuthusto host volatiles and insecticides.AbstractChemosensory protein (CSP) genes significantly enriched in the female antennae are potential molecular candidates for mediating female oviposition behaviors. In this study, we presented the interaction mechanisms of a female-antenna-biased PxutCSP19 inPapilio xuthusto 47 host volatiles, four biopesticides and 24 synthetic insecticides. Using a bioinformatics-based homology search, 22 genes orthologous to PxutCSP19 were identified from 22 otherPapiliobutterflies with high sequence identities to each other (73.20~98.72%). Multiple alignment analyses revealed a particularly extended N-terminus ofPapilioCSP19s (an average of 154 residues) compared to insects’ typical CSPs (approximately 120 residues). The expression profiles indicated that PxutCSP19 was significantly enriched in the female antennae, with a 31.81-fold difference relative to the male antennae. In ligand-binding assays, PxutCSP19 could strongly bind six host odorants with high affinities, ranging from dissociation constant (Ki) values of 20.44 ± 0.64 μM to 22.71 ± 0.73 μM. Notably, this protein was tuned to a monoterpenoid alcohol, linalool, which generally existed in the Rutaceae plants and elicited electrophysiological and behavioral activities of the swallowtail butterfly. On the other hand, PxutCSP19 was also capable of binding eight insecticides with stronger binding abilities (Ki< 12 μM) compared to host odorants. When an extended N-terminal region of PxutCSP19 was truncated into two different proteins, they did not significantly affect the binding of PxutCSP19 to ligands with high affinities, suggesting that this extended N-terminal sequences were not involved in the specificity of ligand recognition. Altogether, our study sheds light on the putative roles of PxutCSP19 enriched in the female antennae ofP. xuthusin the perception of host volatiles and the sequestering of insecticides, and it complements the knowledge of butterfly CSPs in olfaction and insecticide resistance.
研究领域:
昆虫学
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分子生物学
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化学生态学
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PubMed
高影响因子
Neural Regeneration Research
·
2013
·
IF 0.144
Yi Zhang,Shu Zhao,Weiren Dong,Suifen He,Haihong Wang,Lihua Zhang,Yinjuan Tang,Jiasong Guo,Suiqun Guo
The sonic hedgehog protein not only plays a key role in early embryonic development, but also has essential effects on the adult nervous system, including neural stem cell proliferation, differentiation, migration and neuronal axon guidance. The N-terminal fragment of sonic hedgehog is the key functional element in this process. Therefore, this study aimed to clone and analyze the N-terminal fragment of the sonic hedgehog gene. Total RNA was extracted from the notochord of a Sprague-Dawley rat at embryonic day 9 and the N-terminal fragment of sonic hedgehog was amplified by nested reverse transcription-PCR. The N-terminal fragment of the sonic hedgehog gene was successfully cloned. The secondary and tertiary structures of the N-terminal fragment of the sonic hedgehog protein were predicted using Jpred and Phyre online.
研究领域:
分子生物学
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发育生物学
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遗传学
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DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）