Pub Date : 2023-08-08DOI: 10.1007/s10930-023-10143-3
Gloria Narayan, Akriti Agrawal, Plaboni Sen, Shirisha Nagotu, Rajkumar P Thummer
Paired box 4 (PAX4) is a pivotal transcription factor involved in pancreatogenesis during embryogenesis, and in adults, it is key for β-cell proliferation and survival. Additionally, PAX4 also functions as a tumor suppressor protein in human melanomas. The present study demonstrates the production of bioactive recombinant human PAX4 transcription factor. At first, the inserts (PAX4 protein-coding sequence having tags at either ends) were cloned in an expression vector to give rise to pET28a(+)-HTN-PAX4 and pET28a(+)-PAX4-NTH genetic constructs, and these were then transformed into Escherichia coli (E. coli) for their expression. The HTN-PAX4 and PAX4-NTH fusion proteins produced were purified with a yield of ~ 3.15 mg and ~ 0.83 mg, respectively, from 1.2 L E. coli culture. Further, the secondary structure retention of the PAX4 fusion proteins and their potential to internalize the mammalian cell and its nucleus was demonstrated. The bioactivity of these fusion proteins was investigated using various assays (cell migration, cell proliferation and cell cycle assays), demonstrating it to function as a tumor suppressor protein. Thus, this macromolecule can prospectively help understand the function of human PAX4 in cellular processes, disease-specific investigations and direct cellular reprogramming.
{"title":"Production of Bioactive Human PAX4 Protein from E. coli","authors":"Gloria Narayan, Akriti Agrawal, Plaboni Sen, Shirisha Nagotu, Rajkumar P Thummer","doi":"10.1007/s10930-023-10143-3","DOIUrl":"10.1007/s10930-023-10143-3","url":null,"abstract":"<div><p>Paired box 4 (PAX4) is a pivotal transcription factor involved in pancreatogenesis during embryogenesis, and in adults, it is key for β-cell proliferation and survival. Additionally, PAX4 also functions as a tumor suppressor protein in human melanomas. The present study demonstrates the production of bioactive recombinant human PAX4 transcription factor. At first, the inserts (<i>PAX4</i> protein-coding sequence having tags at either ends) were cloned in an expression vector to give rise to pET28a(+)-HTN-PAX4 and pET28a(+)-PAX4-NTH genetic constructs, and these were then transformed into <i>Escherichia coli</i> (<i>E. coli</i>) for their expression. The HTN-PAX4 and PAX4-NTH fusion proteins produced were purified with a yield of ~ 3.15 mg and ~ 0.83 mg, respectively, from 1.2 L <i>E. coli</i> culture. Further, the secondary structure retention of the PAX4 fusion proteins and their potential to internalize the mammalian cell and its nucleus was demonstrated. The bioactivity of these fusion proteins was investigated using various assays (cell migration, cell proliferation and cell cycle assays), demonstrating it to function as a tumor suppressor protein. Thus, this macromolecule can prospectively help understand the function of human PAX4 in cellular processes, disease-specific investigations and direct cellular reprogramming.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2023-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9945842","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Spider venom contains various peptides and proteins, which can be used for pharmacological applications. Finding novel therapeutic strategies against neurodegenerative diseases with the use of purified peptides and proteins, extracted from spiders can be greatly precious. Neurodegenerative diseases are rapidly developing and expanding all over the world. Excitotoxicity is a frequent condition amongst neuro-degenerative disorders. This harmful process is usually induced through hyper-activation of N-Methyl-D-Aspartate (NMDA) receptor, and P/Q-type voltage-gated calcium channels (VGCCs). The omega-agatoxin-Aa4b is a selective and strong VGCCblocker. This study aimed to investigate the effects of this blocker on the NMDA-induced memory and learning defect in rats. For this purpose, nineteen spiders of the funnel-weaver Agelena orientalis species were collected. The extracted venom was lyophilized andpurified through gel-filtration chromatography, and capillary electrophoresis techniques. Subsequently, mass spectrometry (HPLC-ESI-MS) was used for identification of this bio-active small protein. Afterward, the effect of the omega-agatoxin-Aa4b (2 μg, intra-cornu ammonis-3 of the hippocampus) on the NMDA-induced learning and memory deficits in rats was evaluated. Learning and memory performances were evaluated by the use of passive avoidance test. For synaptic quantification and memory function the amount of calcium/calmodulin-dependent protein kinase ІІ (CaCdPKІІ) gene expression was measured using the Real-time PCR technique. To compare the experimental groups, hematoxylin and eosin (H&E) staining of hippocampus tissues was performed. Our results rendered that the omega-Agatoxin-Aa4b treatment can ameliorate and reverse the learning and memory impairment caused by NMDA-induced excitotoxicity in rat hippocampus.
蜘蛛毒液含有多种多肽和蛋白质,可用于药理应用。利用从蜘蛛中提取的纯化肽和蛋白质,寻找对抗神经退行性疾病的新治疗策略是非常宝贵的。神经退行性疾病在世界范围内迅速发展和扩大。兴奋性毒性是神经退行性疾病中的一种常见疾病。这种有害的过程通常是通过n -甲基- d -天冬氨酸(NMDA)受体和P/ q型电压门控钙通道(VGCCs)的过度激活诱导的。omega-agatoxin-Aa4b是一种选择性强效的vgcc阻滞剂。本研究旨在探讨该阻滞剂对nmda诱导的大鼠记忆和学习缺陷的影响。为此,我们收集了19只漏斗织蜘蛛——Agelena orientalis。提取的毒液经冻干、凝胶过滤层析和毛细管电泳纯化。随后,采用质谱法(HPLC-ESI-MS)对该生物活性小蛋白进行鉴定。随后,我们评估了omega-agatoxin-Aa4b (2 μg,海马角内氨-3)对nmda诱导大鼠学习记忆缺陷的影响。采用被动回避测验评估学习记忆能力。对于突触定量和记忆功能,使用Real-time PCR技术测量钙/钙调素依赖性蛋白激酶ІІ (CaCdPKІІ)基因表达量。采用苏木精和伊红(H&E)染色对各组海马组织进行比较。我们的研究结果表明,omega-Agatoxin-Aa4b治疗可以改善和逆转nmda诱导的大鼠海马兴奋性毒性引起的学习和记忆障碍。
{"title":"Purified Native Protein Extracted from the Venom of Agelena orientalis Attenuates Memory Defects in the Rat Model of Glutamate-Induced Excitotoxicity","authors":"Mohammad Keimasi, Kowsar Salehifard, Sayyed Jafar Hoseini, Fariba Esmaeili, Noushin Mirshah Jafar Esfahani, Mohammadreza Amirsadri, Mohammadjavad Keimasi, Majid Moradmand, Mohammad Reza Mofid","doi":"10.1007/s10930-023-10140-6","DOIUrl":"10.1007/s10930-023-10140-6","url":null,"abstract":"<div><p>Spider venom contains various peptides and proteins, which can be used for pharmacological applications. Finding novel therapeutic strategies against neurodegenerative diseases with the use of purified peptides and proteins, extracted from spiders can be greatly precious. Neurodegenerative diseases are rapidly developing and expanding all over the world. Excitotoxicity is a frequent condition amongst neuro-degenerative disorders. This harmful process is usually induced through hyper-activation of N-Methyl-D-Aspartate (NMDA) receptor, and P/Q-type voltage-gated calcium channels (VGCCs). The omega-agatoxin-Aa4b is a selective and strong VGCCblocker. This study aimed to investigate the effects of this blocker on the NMDA-induced memory and learning defect in rats. For this purpose, nineteen spiders of the funnel-weaver <i>Agelena orientalis</i> species were collected. The extracted venom was lyophilized andpurified through gel-filtration chromatography, and capillary electrophoresis techniques. Subsequently, mass spectrometry (HPLC-ESI-MS) was used for identification of this bio-active small protein. Afterward, the effect of the omega-agatoxin-Aa4b (2 μg, intra-cornu ammonis-3 of the hippocampus) on the NMDA-induced learning and memory deficits in rats was evaluated. Learning and memory performances were evaluated by the use of passive avoidance test. For synaptic quantification and memory function the amount of calcium/calmodulin-dependent protein kinase ІІ (CaCdPKІІ) gene expression was measured using the Real-time PCR technique. To compare the experimental groups, hematoxylin and eosin (H&E) staining of hippocampus tissues was performed. Our results rendered that the omega-Agatoxin-Aa4b treatment can ameliorate and reverse the learning and memory impairment caused by NMDA-induced excitotoxicity in rat hippocampus.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2023-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4071942","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-07-25DOI: 10.1007/s10930-023-10139-z
Bingxin Liu, Qiucui Guo, Zong Li, Xiaoxiao Guo, Xinchun Liu
Bacterial biofilms are widespread in the environment, and bacteria in the biofilm are highly resistant to antibiotics and possess host immune defense mechanisms, which can lead to serious clinical and environmental health problems. The increasing problem of bacterial resistance caused by the irrational use of traditional antimicrobial drugs has prompted the search for better and novel antimicrobial substances. In this paper, we review the effects of phage endolysins, modified phage endolysins, and their combination with other substances on bacterial biofilms and provide an outlook on their practical applications. Phage endolysins can specifically and efficiently hydrolyze the cell walls of bacteria, causing bacterial lysis and death. Phage endolysins have shown superior bactericidal effects in vitro and in vivo, and no direct toxicity in humans has been reported to date. The properties of phage endolysins make them promising for the prevention and treatment of bacterial infections. Meanwhile, endolysins have been genetically engineered to exert a stronger scavenging effect on biological membranes when used in combination with antibiotics and drugs. Phage endolysins are powerful weapons for controlling bacterial biofilms.