{"title":"重组人鸢尾素(12.5 kDa)的表达、纯化及生物学特性研究","authors":"Kalpana Panati , Venkata Ramireddy Narala , Vydyanath R. Narasimha , Madhavi Derangula , Venkat R.R. Arva Tatireddigari , Suneetha Yeguvapalli","doi":"10.1016/j.jgeb.2018.06.007","DOIUrl":null,"url":null,"abstract":"<div><p>Fibronectin type III domain containing 5 (FNDC5) is a transmembrane protein. Upon cleavage, it yields a peptide called irisin that is supposedly bind to an unknown receptor and facilitates browning of white adipose tissue (WAT). Increased levels of irisin are associated with increased levels of energy expenditure markers PGC-1α, UCP-1, besides abundance of beige adipocytes in WAT. Though varied sizes of irisin were reported in humans and rodents it is not yet clear about the actual size of the irisin produced physiologically. Hence, we cloned and expressed human irisin (32–143 aa of FNDC5) in <em>Escherichia coli</em> based on the proposed cleavage site that yields 12.5 kDa peptide to study its antigenicity and other biological functions <em>in vitro</em>. We purified recombinant human irisin (rh-irisin) to 95% homogeneity with simple purification method with a yield of 25 mg/g wet cell pellet. rh-irisin has been detected by commercially available antibodies from different sources with similar antigenicity. Biological activity of the rh-irisin was confirmed by using 3T3-L1 pre-adipocyte differentiation by Oil red O staining. Further, rh-irisin treatment on pre-adipocytes showed increased expression of markers associated with energy expenditure. As it is involved in energy expenditure process, it could be considered as potential therapeutic option for various metabolic diseases.</p></div>","PeriodicalId":53463,"journal":{"name":"Journal of Genetic Engineering and Biotechnology","volume":null,"pages":null},"PeriodicalIF":3.5000,"publicationDate":"2018-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jgeb.2018.06.007","citationCount":"10","resultStr":"{\"title\":\"Expression, purification and biological characterisation of recombinant human irisin (12.5 kDa)\",\"authors\":\"Kalpana Panati , Venkata Ramireddy Narala , Vydyanath R. Narasimha , Madhavi Derangula , Venkat R.R. Arva Tatireddigari , Suneetha Yeguvapalli\",\"doi\":\"10.1016/j.jgeb.2018.06.007\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Fibronectin type III domain containing 5 (FNDC5) is a transmembrane protein. Upon cleavage, it yields a peptide called irisin that is supposedly bind to an unknown receptor and facilitates browning of white adipose tissue (WAT). Increased levels of irisin are associated with increased levels of energy expenditure markers PGC-1α, UCP-1, besides abundance of beige adipocytes in WAT. Though varied sizes of irisin were reported in humans and rodents it is not yet clear about the actual size of the irisin produced physiologically. Hence, we cloned and expressed human irisin (32–143 aa of FNDC5) in <em>Escherichia coli</em> based on the proposed cleavage site that yields 12.5 kDa peptide to study its antigenicity and other biological functions <em>in vitro</em>. We purified recombinant human irisin (rh-irisin) to 95% homogeneity with simple purification method with a yield of 25 mg/g wet cell pellet. rh-irisin has been detected by commercially available antibodies from different sources with similar antigenicity. Biological activity of the rh-irisin was confirmed by using 3T3-L1 pre-adipocyte differentiation by Oil red O staining. Further, rh-irisin treatment on pre-adipocytes showed increased expression of markers associated with energy expenditure. As it is involved in energy expenditure process, it could be considered as potential therapeutic option for various metabolic diseases.</p></div>\",\"PeriodicalId\":53463,\"journal\":{\"name\":\"Journal of Genetic Engineering and Biotechnology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.5000,\"publicationDate\":\"2018-12-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1016/j.jgeb.2018.06.007\",\"citationCount\":\"10\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Genetic Engineering and Biotechnology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1687157X18300672\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"Biochemistry, Genetics and Molecular Biology\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Genetic Engineering and Biotechnology","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1687157X18300672","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 10
摘要
纤维连接蛋白III型结构域含有5 (FNDC5)是一种跨膜蛋白。在裂解过程中,它产生一种叫做鸢尾素的肽,这种肽被认为与一种未知的受体结合,并促进白色脂肪组织(WAT)的褐化。鸢尾素水平的升高与WAT中能量消耗标志物PGC-1α、UCP-1水平的升高有关,此外还与米色脂肪细胞的丰富度有关。虽然在人类和啮齿动物中有不同大小的鸢尾素的报道,但尚不清楚生理上产生的鸢尾素的实际大小。因此,我们基于提出的产生12.5 kDa肽的裂解位点,在大肠杆菌中克隆并表达了人鸢尾素(32 - 143aa of FNDC5),并在体外研究了其抗原性和其他生物学功能。我们用简单的纯化方法纯化了重组人鸢尾素(rh-irisin),纯度为95%,湿细胞颗粒的产量为25 mg/g。鸢尾素已被市售的具有相似抗原性的不同来源的抗体检测到。通过3T3-L1脂肪前细胞分化油红O染色证实了鸢尾素的生物活性。此外,rh-鸢尾素对前脂肪细胞的处理显示与能量消耗相关的标志物表达增加。由于它参与能量消耗过程,可以被认为是各种代谢性疾病的潜在治疗选择。
Expression, purification and biological characterisation of recombinant human irisin (12.5 kDa)
Fibronectin type III domain containing 5 (FNDC5) is a transmembrane protein. Upon cleavage, it yields a peptide called irisin that is supposedly bind to an unknown receptor and facilitates browning of white adipose tissue (WAT). Increased levels of irisin are associated with increased levels of energy expenditure markers PGC-1α, UCP-1, besides abundance of beige adipocytes in WAT. Though varied sizes of irisin were reported in humans and rodents it is not yet clear about the actual size of the irisin produced physiologically. Hence, we cloned and expressed human irisin (32–143 aa of FNDC5) in Escherichia coli based on the proposed cleavage site that yields 12.5 kDa peptide to study its antigenicity and other biological functions in vitro. We purified recombinant human irisin (rh-irisin) to 95% homogeneity with simple purification method with a yield of 25 mg/g wet cell pellet. rh-irisin has been detected by commercially available antibodies from different sources with similar antigenicity. Biological activity of the rh-irisin was confirmed by using 3T3-L1 pre-adipocyte differentiation by Oil red O staining. Further, rh-irisin treatment on pre-adipocytes showed increased expression of markers associated with energy expenditure. As it is involved in energy expenditure process, it could be considered as potential therapeutic option for various metabolic diseases.
期刊介绍:
Journal of genetic engineering and biotechnology is devoted to rapid publication of full-length research papers that leads to significant contribution in advancing knowledge in genetic engineering and biotechnology and provide novel perspectives in this research area. JGEB includes all major themes related to genetic engineering and recombinant DNA. The area of interest of JGEB includes but not restricted to: •Plant genetics •Animal genetics •Bacterial enzymes •Agricultural Biotechnology, •Biochemistry, •Biophysics, •Bioinformatics, •Environmental Biotechnology, •Industrial Biotechnology, •Microbial biotechnology, •Medical Biotechnology, •Bioenergy, Biosafety, •Biosecurity, •Bioethics, •GMOS, •Genomic, •Proteomic JGEB accepts