MOLECULES
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2019
·Vol. 24
·期 17
·3146
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IF 3.06
Weigui Luo,Yuan Xiao,Qiwen Liang,Yi Su,Langtao Xiao
Small signaling peptides (SSPs) are a class of short peptides playing critical roles in plant growth and development. SSPs are also involved in the phytohormone signaling pathway. However, identification of mature SSPs is still a technical challenge because of their extremely low concentrations in plant tissue and complicated interference by many other metabolites. Here, we report an optimized protocol to extract SSPs based on protoplast extraction and to analyze SSPs based on tandem mass spectrometry peptidomics. Using plant protoplasts as the material, soluble peptides were directly extracted into phosphate buffer. The interference of non-signaling peptides was significantly decreased. Moreover, we applied the protocol to identify potential SSPs in auxin treated wild type and auxin biosynthesis defective mutantyuc2yuc6. Over 100 potential SSPs showed a response to auxin inArabidopsis thaliana.Keywords:small signaling peptides;plant protoplast;peptidomics;auxin
研究领域:
植物生物学
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分子生物学
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生物化学
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PubMed
高影响因子
Frontiers in Bioengineering and Biotechnology
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2021
·
IF 5.89
Fu Xueqing,Zhang Fangyuan,Ma Yanan,Hassani Danial,Peng Bowen,Pan Qifang,Zhang Yuhua,Deng Zhongxiang,Liu Wenbo,Zhang Jixiu,Han Lei,Chen Dongfang,Zhao Jingya,Li Ling,Sun Xiaofen,Tang Kexuan
Terpenes constitute the largest class of secondary metabolites in plants. Some terpenes are essential for plant growth and development, membrane components, and photosynthesis. Terpenes are also economically useful for industry, agriculture, and pharmaceuticals. However, there is very low content of most terpenes in microbes and plants. Chemical or microbial synthesis of terpenes are often costly. Plants have the elaborate and economic biosynthetic way of producing high-value terpenes through photosynthesis. Here we engineered the heterogenous sesquiterpenoid patchoulol production in A. annua. When using a strong promoter such as 35S to over express the avian farnesyl diphosphate synthase gene and patchoulol synthase gene, the highest content of patchoulol was 52.58 μg/g DW in transgenic plants. When altering the subcellular location of the introduced sesquiterpene synthetase via a signal peptide, the accumulation of patchoulol was observably increased to 273 μg/g DW. This case demonstrates that A. annua plant with glandular trichomes is a useful platform for synthetic biology studies.
研究领域:
植物学
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合成生物学
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药用植物遗传工程
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DOI
PubMed
高影响因子
Nanoscale
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2015
·Vol. 7
·期 14
·6104-6113
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IF 7.394
Lei Yang,Weihua Jiang,Lipeng Qiu,Xuewei Jiang,Daiying Zuo,Dongkai Wang,Li Yang
Strong blue fluorescent polyethylene glycol (PEG) anchored carbon nitride dots (CDs@PEG) with a high quantum yield (QY) of 75.8% have been synthesized by a one step hydrothermal treatment. CDs with a diameter of ca. 6 nm are well dispersed in water and present a graphite-like structure. Photoluminescence (PL) studies reveal that CDs display excitation-dependent behavior and are stable under various test conditions. Based on the as-prepared CDs, we designed novel cell nucleus targeting imaging carbon dots functionalized with a nuclear localization signal (NLS) peptide. The favourable biocompatibilities of CDs and NLS modified CDs (NLS-CDs) are confirmed by in vitro cytotoxicity assays. Importantly, intracellular localization experiments in MCF7 and A549 cells demonstrate that NLS-CDs could be internalized in the nucleus and show blue light, which indicates that CDs may serve as cell nucleus imaging probes.
研究领域:
生物化学
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材料科学
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细胞生物学
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DOI
PubMed
高影响因子
Nanoscale
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2016
·Vol. 8
·期 12
·6801-6809
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IF 7.76
Lei Yang,Zheran Wang,Ju Wang,Weihua Jiang,Xuewei Jiang,Zhaoshi Bai,Yunpeng He,Jianqi Jiang,Dongkai Wang,Li Yang
Carbon dots (CDs) have shown great potential in imaging and drug/gene delivery applications. In this work, CDs functionalized with a nuclear localization signal peptide (NLS-CDs) were employed to transport doxorubicin (DOX) into cancer cells for enhanced antitumor activity. DOX was coupled to NLS-CDs (DOX-CDs) through an acid-labile hydrazone bond, which was cleavable in the weakly acidic intracellular compartments. The cytotoxicity of DOX-CD complexes was evaluated by the MTT assay and the cellular uptake was monitored using flow cytometry and confocal laser scanning microscopy. Cell imaging confirmed that DOX-CDs were mainly located in the nucleus. Furthermore, the complexes could efficiently induce apoptosis in human lung adenocarcinoma A549 cells. The in vivo therapeutic efficacy of DOX-CDs was investigated in an A549 xenograft nude mice model and the complexes exhibited an enhanced ability to inhibit tumor growth compared with free DOX. Thus, the DOX-CD conjugates may be exploited as promising drug delivery vehicles in cancer therapy.
研究领域:
药学
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生物医学工程
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肿瘤学
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DOI
PubMed
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
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2024
·Vol. 108
·期 1
·1-12
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IF 3.9
Chen Qianlin,Wang Bin,Pan Li
Abstract In this study, we successfully applied the strategy of combining tandem promoters and tandem signal peptides with overexpressing signal peptidase to efficiently express and produce γ-glutamyl peptidase (GGT) enzymes (BsGGT, BaGGT, and BlGGT) from Bacillus subtilis , Bacillus amyloliquefaciens , and Bacillus licheniformis in Bacillus subtilis ATCC6051Δ5. In order to avoid the problem of instability caused by duplicated strong promoters, we assembled tandem promoters of different homologous genes from different species. To achieve resistance marker-free enzyme in the food industry, we first removed the replication origin and corresponding resistance marker of Escherichia coli from the expression vector. The plasmid was then transformed into the B. subtilis host, and the Kan resistance gene in the expression plasmid was directly edited and silenced using the CRISPR/Cas9n-AID base editing system. As a result, a recombinant protein expression carrier without resistance markers was constructed, and the enzyme activity of the BlGGT strain during shake flask fermentation can reach 53.65 U/mL. The recombinant BlGGT was immobilized with epoxy resin and maintained 82.8% enzyme activity after repeated use for 10 times and 87.36% enzyme activity after storage at 4 °C for 2 months. The immobilized BlGGT enzyme was used for the continuous synthesis of theanine with a conversion rate of 65.38%. These results indicated that our approach was a promising solution for improving enzyme production efficiency and achieving safe production of enzyme preparations in the food industry. Key points
• Efficient expression of recombinant proteins by a combination of dual promoter and dual signal peptide.
• Construction of small vectors without resistance markers in B. subtilis using CRISPR/Cas9n-AID editing system.
• The process of immobilizing BlGGT with epoxy resin was optimized.
研究领域:
微生物学
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食品科学
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分子生物学
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2022
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IF 5.005
Qi Shen,Xiao-Ting Zhou,Qian Guo,Yu-Zhen Xue,Ya-Ping Xue,Yu-Guo Zheng
Pichia pastoris is widely used for the production of valuable recombinant proteins. An advantage of P. pastoris over other expression systems is that it secretes low levels of endogenous proteins, which facilitates the purification processes if the desired recombinant proteins are efficiently secreted into the culture medium. However, not all recombinant proteins can be successfully secreted by P. pastoris, especially enzymes that are located in intracellular compartments in their native hosts. Few studies have reported strategies for releasing recombinant proteins which cannot be secreted by standard protocols. Here, we investigated whether this challenge can be addressed using novel secretion leaders. Analysis of the secretome and transcriptome of P. pastoris indicated that the four genes with the highest protein-to-transcript ratios were EPX1, PAS_chr3_0030, SCW10, and UTH1, suggesting that their gene products contain efficient secretion leaders. Our data revealed that the signal peptide derived from the PAS_chr3_0030 gene product conferred secretion competence to certain industrial enzymes, e.g., a nitrilase of Alcaligenes faecalis ZJUTB10, a ribosylnicotinamide kinase of P. pastoris, and a glucose dehydrogenase of Exiguobacterium sibiricum. Therefore, the signal peptide derived from the PAS_chr3_0030 gene product represents a novel secretion sequence for the secretory expression of recombinant enzymes in P. pastoris.IMPORTANCE Although P. pastoris is widely used for the secretory production of pharmaceutical proteins, its successful applications in the secretory production of industrial enzymes are limited. The α-mating factor pre-pro leader is the most widely used secretion signal in P. pastoris, but numerous industrial enzymes cannot be secreted using it. The importance of this study is that we identified a signal peptide derived from the PAS_chr3_0030 gene product which conferred secretion competence to three-quarters of the enzymes tested. This signal peptide derived from the PAS_chr3_0030 gene product may facilitate the application of P. pastoris in industrial biocatalysis.
研究领域:
分子生物学
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生物技术
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蛋白质工程
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酶学
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DOI
PubMed
高影响因子
Frontiers in Bioengineering and Biotechnology
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2021
·
IF 5.89
Miao Ting,Basit Abdul,Liu Junquan,Zheng Fengzhen,Rahim Kashif,Lou Huiqiang,Jiang Wei
Xylanases with high specific activity has been focused with great interest as a useful enzyme in biomass utilization. The production of recombinant GH11 xylanase (MYCTH_56237) from Myceliophthora thermophila has been improved through N-terminal signal peptide engineering in P. pastoris. The production of newly recombinant xylanase (termed Mtxyn11C) was improved from 442.53 to 490.7 U/mL, through a replacement of α-factor signal peptide with the native xylanase signal peptide segment (MVSVKAVLLLGAAGTTLA) in P. pastoris. Scaling up of Mtxyn11C production in a 7.5 L fermentor was improved to the maximal production rate of 2503 U/mL. In this study, the degradation efficiency of Mtxyn11C was further examined. Analysis of the hydrolytic mode of action towards the birchwood xylan (BWX) revealed that Mtxyn11C was clearly more effective than commercial xylanase and degrades xylan into xylooligosaccharides (xylobiose, xylotriose, xylotetraose). More importantly, Mtxyn11C in combination with a single multifunctional xylanolytic enzyme, improved the hydrolysis of BWX into single xylose by 40%. Altogether, this study provided strategies for improved production of xylanase together with rapid conversion of xylose from BWX, which provides sustainable, cost-effective and environmental friendly approaches to produce xylose/XOSs for biomass energy or biofuels production.
研究领域:
生物技术
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酶学
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生物化学工程
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PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Suifei Li,Junyang Zhong,Yunxiao Ma,Chengfeng Yue,Wenxia Lv,Guodong Ye,Xiumei Tian,Xin Li,Yugang Huang,Lingran Du
Transcriptional gene silencing (TGS) mediated by siRNA holds promise for long-term silencing efficacy, determined by effective nuclear delivery of siRNA. However, non-viral vectors for this purpose are limited. In this work, we synthesized guanidinylated linear synthetic polypeptides (GLSPs) to explore how chain length and conformation impact siRNA delivery, especially nuclear entry. Results show that helical conformations, particularly right-handed ones, enhance siRNA loading and silencing efficiency compared to unordered structures. Increasing chain length also improves these aspects. The endocytic pathways of carrier/siRNA nanocomplexes (NCs) are mainly determined by conformation, regardless of length. Notably, some NCs derived from right-handed helices can enter cells via direct membrane penetration, like bioactivity of cell penetrating peptides (CPPs). When the peptide chain of GLSPs is long enough, all vectors can rapidly deliver siRNA to the nucleus, similar to bioactivity of nuclear localization signal peptides (NLSPs). Interestingly, helicity of the vectors aids endosomal escape of NCs. Moreover, delivering siRNA to the nucleus via GLSPs induces TGS associated with DNA promoter methylation or histone deacetylation. This study clarifies the structure-activity relationship of GLSPs in siRNA delivery, providing new insights for designing non-viral carriers for TGS.
研究领域:
分子生物学
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基因治疗
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药物递送系统
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PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
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2022
·
IF 4.446
He Li,Dongbang Yao,Jingru Ying,Xudong Han,Xuecheng Zhang,Xiuju Fang,Zemin Fang,Yazhong Xiao
A strategy that combines signal peptide (SP) optimization and translation efficiency improvement was used to enhance the extracellular production of raw starch-degrading α-amylase AmyZ1 from Pontibacillus sp. ZY in Bacillus subtilis . The optimal SP YpuA screened from 173 B. subtilis SPs mediated a 1.28-fold higher AmyZ1 activity than the original SP amyQ . Then, strategies of translation efficiency improvement, including optimizing the Shine-Dalgarno-like (SD-like) sequence, constructing a dual 5′-untranslated region (dual-UTR), optimizing the spacer region of the UTR, and optimizing the 5′-proximal coding sequence, were used to enhance AmyZ1 production. The resulting recombinant strain (BZYACO6) containing SD-like3, dUTR2, and an optimized 5′-proximal coding sequence (O6) produced the highest extracellular α-amylase activity of 2974.0 U/mL in shake flask, 2.07-fold higher than that of the original strain BZd34 (1437.6 U/mL). After fermentation optimization, the BZYACO6 strain produced an extracellular AmyZ1 activity of 3915.2 U/mL in shake flask and 25,070 U/mL in 3-L fermenter, representing the highest level of recombinant raw starch-degrading α-amylase production reported to date. Furthermore, this work provides a strategic reference for enhancing the extracellular production of other recombinant proteins in B. subtilis .
研究领域:
微生物学
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生物技术
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酶学
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DOI
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2019
·
IF 4.784
Xingyu Lu,Qian Zhong,Jian Liu,Fulin Yang,Chenghui Lu,Huan Xiong,Sha Li,Yibo Zhu,Lingtian Wu
Chondroitinase ABC I (ChSase ABC I) is a key enzyme of chondroitin sulfate (CS) degradation and widely used for CS detection in the medicine filed. However, the recombinant ChSase ABC I was weakly expressed in Escherichia coli because the forms of it were mostly inclusion bodies. In this study, a signal peptide ( pel B) was used for the soluble form expression of ChSase ABC I in E. coli . Then the culture condition for ChSase ABC I expression was optimized through response surface methodology. Results revealed that the expression level of ChSase ABC I in a 7.5 L fermentor (29.03 mL −1 ) was approximately 1.65-fold higher than that of the shake flask level (17.55 mL −1 ). The enzymatic properties and kinetic constants of recombinant ChSase ABC I were also studied. Recombinant ChSase ABC I was also used to detect the specific disaccharides content of CS from different sources. This study not only eliminates the problem of the enzyme expressed as an inclusion body, but also solves the current problem of expensive ChSase ABC. In a word, it would be an ideal strategy for ChSase ABC high-efficiency expression and a great method to detect specific disaccharides of CS in biomedical field.
研究领域:
生物技术
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医学
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酶学
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DOI
PubMed
高影响因子
BIOTECHNOLOGY LETTERS
·
2022
·Vol. 45
·期 1
·115-124
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IF 2.716
Jin Jing-Hao,Zhou Xue,Liu Wang,Zhang Zi-Hui,Huang Shen-Xin,Zhao Wen-Jing,Chen Xiao-Ren
Objective To examine the influence of widely used protein affinity tags and the tobacco PR1a signal peptide (SP) on detection, purification and bioactivity analyses of the small oomycete apoplastic effector SCR96 in planta . Results Through agroinfiltration, the phytotoxic effector SCR96 of Phytophthora cactorum was expressed in Nicotiana benthamiana leaf apoplast as a fusion protein carrying single affinity tag (His, HA or FLAG) at either C- or N-terminus. Leaf necrosis caused by different affinity-tagged SCR96 varied among tags and replicates. All of tagged proteins can be detected by antibodies against SCR96. All of SCR96 fusions except N-terminally fused 6His-tagged protein were detected using tag antibodies, indicating that 6His tag may be degraded when fused at N-terminus. Interestingly, C-terminal His- and FLAG-tagged SCR96 maintained the biological activity after purification. In the substitution assay of SCR96 SP, we observed that PR1a SP can lead chimeric SCR96 expression in N. benthamiana , but the replacement totally disrupted its bioactivity. Conclusion C-terminal His or FLAG tag, along with its original SP, is efficient enough to enable detection and purification of functional SCR96 from N. benthamiana leaf apoplast, which would facilitate plant–pathogen interaction studies.
研究领域:
植物病理学
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分子生物学
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蛋白质工程
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DOI
PubMed
高影响因子
BIORESOURCE TECHNOLOGY
·
2023
·
IF 11.4
Lizhu Aer,Huiling Qin,Peng Wo,Juan Feng,Lixia Tang
The use of polyethylene terephthalate (PET) results in a significant amount of plastic waste, which poses a threat to the environment and human health. Dual-enzyme system is promising candidate for PET depolymerization. However, its production in Escherichia coli is challenging, especially for secretory expression. Herein, a novel bifunctional dual-enzyme, TfH-FPE, was constructed through fusion of FAST-PETase and TfH. TfH modifies cell membrane permeability via phospholipid degradation, thus facilitating the secretion of TfH-FPE into the medium. After systematic optimization, purified secreted TfH-FPE reached 104 ± 5.2 mg/L, which is 32.5-fold higher than that of the secreted enzyme using a signal peptide. TfH-FPE exhibits remarkable PET depolymerization capacity compared to FAST-PETase, releasing 6-fold more product than FAST-PETase and 2-fold more product than an equimolar enzyme mixture. Collectively, this study explores a novel secretory approach for efficient production of TfH-FPE and provides a valuable tool to promote PET bio-depolymerization via multi-enzyme cascades.
研究领域:
环境科学
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生物技术
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生物化学
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微生物学
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DOI
PubMed
高影响因子
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