Frontiers in Microbiology
·
2021
·
IF 5.64
Zou Lei,Li Sha,Li Nan,Ruan Shi-Long,Chen Jing,Wu Jing,Yan Dazhong,Chao Hong-Jun
Escherichia coli has been developed as the most common host for recombinant protein expression. Unfortunately, there are still some proteins that are resistant to high levels of heterologous soluble expression in E. coli. Protein and peptide fusion tags are one of the most important methods for increasing target protein expression and seem to influence the expression efficiency and solubility as well. In this study, we identify a short 15-residue enhancing solubility peptide, the PCDS (protocatechuate 3,4-dioxygenase solubility) tag, which enhances heterologous protein expression in E. coli. This PCDS tag is a 45-bp long sequence encoding a peptide tag involved in the soluble expression of protocatechuate 3,4-dioxygenase, encoded by the pcaHG98 genes of Pseudomonas putida NCIMB 9866. The 45-bp sequence was also beneficial for pcaHG98 gene amplification. This tag was shown to be necessary for the heterologous soluble expression of PcaHG98 in E. coli. Purified His6-PcaHG98e04-PCDS exhibited an activity of 205.63±14.23U/mg against protocatechuate as a substrate, and this activity was not affected by a PCDS tag. This PCDS tag has been fused to the mammalian yellow fluorescent protein (YFP) to construct YFP-PCDS without its termination codons and YFPt-PCDS with. The total protein expressions of YFP-PCDS and YFPt-PCDS were significantly amplified up to 1.6-fold and 2-fold, respectively, compared to YFP alone. Accordingly, His6-YFP-PCDS and His6-YFPt-PCDS had 1.6-fold and 3-fold higher soluble protein yields, respectively, than His6-YFP expressed under the same conditions. His6-YFP, His6-YFP-PCDS, and His6-YFPt-PCDS also showed consistent fluorescence emission spectra, with a peak at 530nm over a scanning range from 400 to 700nm. These results indicated that the use of the PCDS tag is an effective way to improve heterologous protein expression in E. coli.
研究领域:
分子生物学
·
蛋白质工程
·
合成生物学
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DOI
PubMed
高影响因子
PHARMACOGENOMICS
·
2020
·
IF 2.339
Keguang Chen,Ruichen Guo,Chunmin Wei
Aim: To evaluate whether the synonymous mutant rs2515641 could affect cytochrome P450 2E1 (CYP2E1) expression and the response to acetaminophen (APAP) or triptolide (TP) treatment. Materials & methods: HepG2 cells were transfected with lentiviral vector containing either CYP2E1-1263C or CYP2E1-1263T. Some of these recombinant cells were then treated with APAP or TP. CYP2E1 gene expression was detected by PCR and western blot. Results:CYP2E1 gene expression decreased significantly both in mRNA and protein level after rs2515641 mutation, indicating that this polymorphism can affect both transcription and translation. Furthermore, rs2515641 mutation dramatically changes the response of CYP2E1 expression to APAP or TP treatment. Conclusion: Rs2515641 significantly changes CYP2E1 expression and function, which would be expected to affect drug disposition and response.
研究领域:
药理学
·
分子生物学
·
遗传学
·
毒理学
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DOI
PubMed
高影响因子
PROTEIN EXPRESSION AND PURIFICATION
·
2015
·
IF 1.695
Xin Chen,Yong-hao Hu,Wei-di Chen,Wen-di Li,Zi-chao Huang,Yun Li,Yu-wen Luo,Yu-xia Huang,Yi-tai Chen,Kai Wang,Ling Li
Background Plectasin might serve as a substitute for traditional antibiotics, but its yields and antimicrobial activities warrant further investigation. Objective To identify the influence of inducible versus constitutive expression of plectasin on yields and antimicrobial activities. Methods Through SOE-PCR, a recombinant plectasin gene was generated and inserted into inducible (pPICZαA) and constitutive (pGAPZαA) vectors in order to create Pichia pastori s GS115 strains. After 120 h of fermentation, supernatants were purified by an AKTA purifier using nickel columns. Minimal inhibitory concentration (MIC) and inhibition zone assays were performed after Tricine-SDS-PAGE. Results After 120 h of fermentation, the yield of constitutive plectasin (370 μg/ml) was much lower than that from inducible vector (880 μg/ml) ( P < 0.05). However, constitutive strain reached its plateau phase faster and keep more consistent yield ( P < 0.05). The MICs of inducible plectasin against Methicillin-resistant Staphylococcus aureus (MRSA) 15471118, vancomycin-resistant Enterococcus feces (VREF), and penicillin-resistant Streptococcus pneumonia (PRSP) 31355 were 64, 32, and 64 μg/ml, respectively, while those of constitutive plectasin were 4, 4, and 16 μg/ml. No significant differences were observed in antimicrobial activities between inducible and constitutive plectasin for MRSA 15471118, VREF and PRSP 31355 (all P ＞ 0.05). However, constitutive plectasin had a larger inhibition zone than inducible plectasin with the same mass. Conclusions Although P. pastoris GS115 (pGAPZαA-Plectasin-GS115) had lower expression than P. pastoris GS115 (pPICZαA-plectasin-GS115), it reached the plateau phase faster, had steadier yields and showed superiority in antimicrobial activities. Therefore, pGAPZαA might be more suitable for expression of plectasin in GS115 compared with pPICZαA.
研究领域:
分子生物学
·
微生物学
·
生物技术
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产品关联
DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2024
·Vol. 91
·期 1
·
IF 3.9
Ji Zeng,Hao Wang,Yuxi Xu,Jianying Han,Yannan Li,Shu'an Wen,Changbu Wu,Dani Li,Zheng Liu,Xiaokang Zhang,Guo-Bao Tian,Min Dong
Clostridioides difficile is an obligate anaerobic, Gram-positive bacterium that produces toxins. Despite technological progress, conducting gene expression analysis of C. difficile under different conditions continues to be labor-intensive. Therefore, there is a demand for simplified tools to investigate the transcriptional and translational regulation of C. difficile. The cell-free gene expression (CFE) system has demonstrated utility in various applications, including prototyping, protein production, and in vitro screening. In this study, we developed a C. difficile CFE system capable of in vitro transcription and translation (TX-TL) in the presence of oxygen. Through optimization of cell extract preparation and reaction systems, we increased the protein yield significantly. Furthermore, our observations indicated that this system exhibited higher protein yield using linear DNA templates than circular plasmids for in vitro expression. The prototyping capability of the C. difficile CFE system was assessed using a series of synthetic Clostridium promoters, demonstrating a good correlation between in vivo and in vitro expression. Additionally, we tested the expression of tcdB and tcdR from clinically relevant C. difficile strains using the CFE system, confirming higher toxin expression of the hypervirulent strain R20291. We believe that the CFE system can not only serve as a platform for in vitro protein synthesis and genetic part prototyping but also has the potential to be a simplified model for studying metabolic regulations in Clostridioides difficile.
研究领域:
微生物学
·
分子生物学
·
生物技术
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DOI
高影响因子
Experimental and Therapeutic Medicine
·
2024
·Vol. 27
·期 5
·1-8
·
IF 2.4
Yunyan Tang,Hongfang Li,Jianhua Tang,Lei Hu,Feifei Ma,Yanmiao Liu,Fushan Tang
Total saikosaponins (TSS) form a group of chemically and biologically active components that can be extracted from Bupleurum, with reported antidepressive, anti‑inflammatory, antiviral, antiendotoxin, antitumor, anti‑pulmonary fibrosis and anti‑gastric ulcer effects. Bupleurum or TSS is frequently utilized in clinical practice alongside other medications (such as entecavir, lamivudine, compound paracetamol and amantadine hydrochloride capsules), leading to an increased risk of drug‑drug interactions. The cytochrome P450 (CYP) family serves a critical role in the metabolism of numerous essential drugs (such as tamoxifen, ibuprofen and phenytoin), where the majority of drug interactions involve CYP‑mediated metabolism. It is therefore essential to understand the effects of key components of Bupleurum on CYPs when administering combination therapies containing TSS or Bupleurum. The present study aimed to investigate the effects of TSS on the mRNA and protein expression of CYP3A4 and CYP1A2 in HepaRG cells. The effects of TSS on the survival of HepaRG cells was investigated using the Cell Counting Kit‑8 (CCK‑8) method. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot (WB) analysis were used to assess the effects of different concentrations of TSS (0, 5, 10 and 15 µg/ml) on CYP3A4 and CYP1A2 mRNA and protein expression in HepaRG cells. Based on the CCK‑8 assay results, it was observed that the cell viability remained above 80% when treated with 1, 5, 10 and 15 µg/ml TSS. Although there was a statistically significant reduced cell viability at TSS concentrations of 10 and 15 µg/ml compared with the control group, the findings indicated that TSS did not exhibit notable cytotoxic effects at these concentrations. Furthermore, RT‑qPCR results revealed that compared with those in the control group, TSS at concentrations of 10 and 15 µg/ml reduced CYP3A4 mRNA expression but increased CYP1A2 mRNA expression in HepaRG cells at concentrations of 15 µg/ml. WB analysis found that TSS at concentrations of 10 and 15 µg/ml downregulated CYP3A4 protein expression in HepaRG cells while increasing CYP1A2 protein expression at concentrations of 15 µg/ml. Results in the present study suggest that TSS can inhibit CYP3A4 mRNA and protein expression, but exerts opposite effects on their CYP1A2 counterparts. These findings suggest that it is necessary to consider drug interactions between clinical preparations containing TSS or Bupleurum and drugs metabolized by CYP3A4 and CYP1A2 to avoid potential adverse drug reactions in clinical practice.
研究领域:
药理学
·
分子生物学
·
毒理学
·
药物代谢
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产品关联
DOI
PubMed
高影响因子
Drug Design Development and Therapy
·
2020
·
IF 3.216
Hongfang Li,Yunyan Tang,Yang Wang,Weipeng Wei,Chengchen Yin,Fushang Tang
Background Bupleurum is one of the most important traditional Chinese medicines and an ingredient in many compound preparations. It is widely used together with other drugs in clinical practice, and thus there is great potential for drug–drug interactions. Saikosaponin D (SsD) is a major bioactive triterpenoid saponin extracted from Bupleurum with anti-inflammatory, anticancer, antioxidative, and antihepatic fibrosis effects. Effects of the main components of Bupleurum on cytochromes P450 (CYPs) need to be clarified in the clinical application of combination therapies of formulations containing SsD or Bupleurum . Purpose This study aimed to investigate the effects of SsD on the CYP1A2 and CYP2D6 mRNAs, protein expression, and relative enzyme activities in HepaRG cells. Methods HepaRG cells were cultured with SsD at concentrations of 0.5, 1, 5 and 10 μM for 72 hours. mRNA and protein expression of CYP1A2 and CYP2D6 were analyzed with real-time PCR and Western blot analysis. Relative enzyme activities were analyzed with HPLC based on consumption of the specific probe substrate. Results SsD significantly induced expression of mRNA and increased relative activity of CYP1A2 in HepaRG cells after the cells had been treated with SsD at concentrations of 1, 5 and 10 μM. SsD also induced protein expression of CYP1A2 at concentrations of 5 and 10 μM. SsD exhibited an inductive effect on CYP2D6 mRNA and protein expression, while increasing the relative activity of CYP2D6 at concentrations of 5 and 10 μM. Conclusion This study is the first to investigate the effect of SsD on CYP1A2 and CYP2D6 in HepaRG cells, and the results may provide some useful information on potential drug–drug interactions related to clinical preparations containing SsD or Bupleurum .
研究领域:
药理学
·
毒理学
·
分子生物学
·
中药学
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产品关联
DOI
PubMed
高影响因子
Molecular Medicine Reports
·
2016
·Vol. 14
·期 4
·3871-3877
·
IF 1.559
Jing Qin,Yongmei Hua
Periodontal ligament cells (PDLCs) are important in homeostasis and remodeling in the mechanically‑stimulated periodontium. The aim of the present study was to investigate the effects of hydrogen sulfide (H2S) on periodontal tissue remodeling by examining the mRNA and protein expression levels of alkaline phosphatase (ALP), osteocalcin (OCN) and collagen type I (COL‑1) in human (h)PDLCs induced by tension force application. Cultured hPDLCs were treated with H2S for 24 h, followed by application of a tension force for 1, 3 and 6 h. Cell proliferation and apoptosis were determined using a Cell Counting Kit 8 assay and flow cytometric analysis, respectively. The mRNA expression levels of ALP, OCN and COL‑1 were quantified using reverse transcription‑quantitative polymerase chain reaction analysis, and western blot analysis was used to detect the protein levels of ALP, OCN and COL‑1. The results demonstrated that the mRNA and protein expression levels of ALP, OCN and COL‑1 increased with H2S treatment in a concentration‑dependent manner, which was enhanced by the application of tension force in a relatively short period of time. These findings suggested that H2S may be important in periodontal tissue remodeling during orthodontic tooth movement via increasing hPDLC differentiation, tissue mineralization, bone formation and collagen synthesis.
研究领域:
牙科研究
·
细胞生物学
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分子生物学
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DOI
PubMed
高影响因子
Frontiers in Plant Science
·
2025
·
IF 4.8
Zhangting Xu,Guihua Zhang,Feixiong Zheng,Xiaoji Deng,Yiming Sun,Jaime A Teixeira da Silva,Xiaoxia Shen,Zhenming Yu
Introduction Dendrobium is a multi-purpose medicinal orchid that grows on the edge of highaltitude cliffs. The content of water-soluble polysaccharides (WSPs), which primarily play a pharmacological role, is the main criterion for evaluating the quality of Dendrobium orchid. Therefore, it is necessary to study the regulatory manner involved in the accumulation of WSPs. Methods D. officinale were treated with methyl jasmonate (MeJA), and WSPs content was measured at different time points to assess the dynamic accumulation pattern. To clarify the role of one finger (Dof) family genes in the MeJA-mediated WSP metabolic pathway, a bioinformatics analysis identified Dof members in D. huoshanense , D. nobile and D. officinale . Based on expression patterns and co-expression analysis, a regulatory factor, DoDof4 was identified. Results In this study, the elicitation of D. officinale by methyl jasmonate (MeJA) increased WSP production, which was further amplified by extending the treatment period. Analysis of transcriptomic data revealed that members of the DNA-binding with Dof gene family members accounted for 4% of all differentially expressed genes coding for transcription factors, following MeJA induction. To clarify the role of Dof family genes in the MeJA-mediated WSP metabolic pathway, a bioinformatics analysis identified 29, 29 and 22 Dof members in D. huoshanense , D. nobile and D. officinale , respectively and these were divided into four groups. DoDof4 was encoded a 31.16 kDa protein composed of 292 amino acids, and was targeted on chromosome 3. Furthermore, DoDof4 was a typical transcription factor that localized in the nucleus, displayed transcriptional activity and increased the WSPs accumulation. DoDof4 was co-expressed with 15 genes involved in the WSP metabolic pathway, eight of which displayed a positive Pearson's correlation coefficient. Additionally, correlation analysis revealed the possible downstream targets ( KM980199 and KP203853 ) of DoDof4 . Discussion The results of the present study suggest that DoDof4 acts as an important regulator in the WSPs metabolic pathway, exhibiting potential values for the improvement of WSPs in Dendrobium species.
研究领域:
植物学
·
分子生物学
·
遗传学
·
生药学
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植物生理学
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产品关联
DOI
PubMed
高影响因子
PHARMAZIE
·
2018
·Vol. 73
·期 8
·465-468
·
IF 1.016
Ma Wenxue,Yao Yawen,Gu Yuyan,Li Fang,Cai Jiaxuan,He Baozhu,Chen Qiuyan,Fang Jian,Ouyang Zizhang
Acute phase protein pentraxin 3 (PTX3) is increased in patients with atherosclerotic cardiovascular diseases, such as acute coronary syndrome and coronary artery vulnerability, thereby serving as an important biomarker for the prognosis of cardiovascular diseases. This study was designed
to evaluate the effects of statins on the expression of PTX3 in human endothelial cells. We observed that PTX3 is a TNFα inducible protein in human endothelial cells. In contrast, treatment with simvastatin reduces basal as well as TNFα induced gene and protein expression of PTX3
in a dose- and time-dependent manner. Our study suggests that reducing PTX3 expression by statins could contribute to its protective effects against endothelial dysfunction and coronary artery plaque destabilization.
研究领域:
心血管疾病
·
药理学
·
分子生物学
✓ 已验证
DOI
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2020
·Vol. 104
·期 16
·7079-7091
·
IF 3.53
Wang Shengli,Han Jing,Xia Jiale,Hu Yanru,Shi Liang,Ren Ang,Zhu Jing,Zhao Mingwen
Abstract Identifying new and economical means to utilize diverse lignocellulosic biomass is an urgent task. Ganoderma lucidum is a well-known edible and medicinal basidiomycete with an excellent ability to degrade a wide range of cellulosic biomass, and its nutrient use efficiency is closely related to the activity of extracellular cellulase. Intracellular nicotinamide adenine dinucleotide (NAD + ) biosynthesis is controlled in response to nutritional status, and NAD + is an essential metabolite involved in diverse cellular processes. Nicotinamide mononucleotide adenylyltransferase (NMNAT) is a common enzyme in three NAD + synthesis pathways. In this study, a homologous gene of nmnat was cloned from G. lucidum and two G. lucidum overexpression strains, OE::nmnat4 and OE::nmnat19, were constructed using an Agrobacterium tumefaciens -mediated transformation method. The G. lucidum nmnat overexpression strains showed obviously increased colony growth on different carbon sources, and intracellular Ca 2+ concentrations in the G. lucidum OE::nmnat4 and OE::nmnat19 strains were increased by 2.04- and 2.30-fold, respectively, compared with those in the wild-type (WT) strains. In the G. lucidum OE::nmnat4 and OE::nmnat19 strains, endo- β -glucanase (CMCase) activity increased by approximately 2.8- and 3-fold, while β -glucosidase ( p NPGase) activity increased by approximately 1.9- and 2.1-fold, respectively, compared with the activity in the WT strains. Furthermore, overexpression of NAD + synthesis pathways was found to elicit cellulase production by increasing the intracellular Ca 2+ concentration. In summary, this study is the first to demonstrate that increased intracellular NAD + contents through overexpression of the nmnat gene of NAD + synthesis pathways may increase cellulase production by increasing intracellular Ca 2+ concentrations in G. lucidum . Key points
The concentration of NAD
+
influences cellulase production in G. lucidum.
The concentration of NAD
+
influences the intracellular Ca
2+
concentration in G. lucidum.
The concentration of NAD
+
influences cellulase production by eliciting a change in intracellular Ca
2+
in G. lucidum.
研究领域:
微生物学
·
生物技术
·
酶学
✓ 已验证
DOI
PubMed
高影响因子
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
·
分子生物学
·
蛋白质表达与纯化
·
结构生物学
·
生物催化
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产品关联
DOI
PubMed
高影响因子
CARBOHYDRATE POLYMERS
·
2014
·
IF 3.916
Jingfen Han,Jia Cai,Wuyinga Borjihan,Tsogzolmaa Ganbold,Tariq M. Rana,Huricha Baigude
RNA interference (RNAi) down-regulates gene expression post-transcriptionally, which is a therapeutically significant phenomenon that could potentially reduce the level of disease related proteins that are undruggable by conventional small molecular approaches. However, clinical application of small interference RNAs (siRNAs) requires design of potent siRNA sequences and development of safe and efficient delivery systems. To create a biocompatible siRNA delivery agent, we chemically modified natural polysaccharide curdlan in a regioselective manner to introduce amino group in the glucose units. The resulting 6-amino-curdlan (6AC) is water soluble and forms nanoparticles upon complexing with siRNAs. The novel curdlan-based nanoparticles efficiently delivered siRNAs to human cancer cells and mouse primary cells, and reduced 70–90% of target mRNA level. Moreover, 6AC nanoparticles delivered siRNA targeting eGFP to mouse embryonic stem (mES) cells stably expressing eGFP, and produced substantial reductions of GFP protein level. The novel curdlan-based nanoparticle is a promising vehicle for delivery of short RNAs to knock down endogenous mRNAs.
研究领域:
生物技术
·
分子生物学
·
药物递送
✓ 已验证
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）