共查询到20条相似文献,搜索用时 15 毫秒
1.
Li A Crimmins DL Luo Q Hartupee J Landt Y Ladenson JH Wilson D Anant S Dieckgraefe BK 《Protein expression and purification》2003,31(2):197-206
Human regenerating (Reg) gene products are regionally expressed by gut-derived tissues, and are markedly up-regulated in cancer and in diseases characterized by mucosal injury. We recently identified Reg IV, a novel regenerating gene product that is uniquely expressed by the normal distal gastrointestinal mucosa. The function remains poorly understood due to the lack of significant purified Reg IV for biochemical and functional studies. Recombinant human Reg IV was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris using the MutS strain KM71H. We describe the unique conditions that are required for efficient production of Reg IV protein in high density fermentation. Optimal protein expression was obtained by reduction of the fermentation temperature and addition of casamino acids as a supplemental nitrogen source and to minimize the activity of yeast produced proteases. Recombinant Reg IV protein was purified by tangential flow filtration and reverse phase chromatography. The purified protein was characterized by amino terminus sequence analysis and MALDI-TOFMS showing that the engineered protein had the expected sequence and molecular weight without secondary modification. Recombinant Reg IV was further characterized by specific monoclonal and polyclonal reagents that function for Western blot analysis and for immunolocalization studies. 相似文献
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Guoyong Hu Jiaqin Shen Li Cheng Di Xiang Zhonghui Zhang Miao He Huili Lu Shunying Zhu Mingyuan Wu Yan Yu Xingpeng Wang Wei Han 《Protein expression and purification》2010,69(2):186-190
Regenerating gene (Reg) IV is a newly discovered member of the regenerating gene family belonging to the calcium (C-type) dependent lectin superfamily. Reg IV is highly expressed in the gastrointestinal tract and markedly up-regulated in colon adenocarcinoma, pancreatic cancer, gastric adenocarcinoma, and inflammatory bowel disease. However, the physiological and pathological functions of Reg IV are largely unknown, partly due to the limited access of the bioactive protein. We report here the first expression and purification of Reg IV proteins using a prokaryotic system. Human Reg IV was expressed in Escherichia coli as an insoluble protein which was identified in the fraction of inclusion body after ultrasonication of the bacteria. After the protein aggregate was solubilized by guanidine–HCl, it was refolded by sucrose and arginine-assisted procedures and purified using cation-exchange chromatography. The protein identity and purity of the final preparation were confirmed by analysis of the protein mass and immune specificity in SDS–PAGE, Western blotting, and HPLC assay. The biological activity of the protein was determined by the HCT116 and HT29 cell proliferation assays. The highly purified bioactive human Reg IV should aid in further characterization of its physiological and pathological functions. 相似文献
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ABSTRACT: Aim The aberrant expression of regenerating islet-derived family member, 4 (Reg IV) has been found in various human cancers. However, the roles of Reg IV gene and its encoding product in human glioma have not been clearly understood. Therefore, the aim of this study was to investigate the clinicopathological significance of Reg IV expression in glioma. METHODS: Reg IV mRNA and protein expression in human gliomas and non-neoplastic brain tissues were respectively detected by real-time quantitative RT-PCR assay, Western blot, and immunohistochemistry. The association of Reg IV immunostaining with clinicopathological factors and prognosis of glioma patients was also statistically analyzed. RESULTS: Reg IV mRNA and protein expression levels in glioma tissues were both significantly higher than those in the corresponding non-neoplastic brain tissues (both P?0.001). Additionally, the increased Reg IV immunostaining in glioma tissues was significantly associated with advanced pathological grade (P?=?0.008). Reg IV protein up-regulation was also significantly correlated with low Karnofsky performance score (KPS) (P?=?0.02). Moreover, the overall survival of patients with high Reg IV protein expression was dramatically shorter than those with low Reg IV protein expression (P?0.001). Multivariate Cox regression analysis further confirmed that Reg IV expression was an independent prognostic factor for patients with gliomas (P?=?0.008). CONCLUSIONS: These convinced evidences suggest for the first time that Reg IV might accelerate disease progression and act as a candidate prognostic marker for gliomas. Virtual slides The virtual slide(s) for this article can be found here: 相似文献
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Wu H Zheng Q Rengabhashyam P Zenilman ME 《The Einstein quarterly journal of biology and medicine》2000,17(4):178
Although the pancreatic regenerating (reg) gene, was first isolated from a rat regenerating islets in 1988, its protein product was originally described in the 1970s. Reg proteins arise from a multigene family with three subtypes, and have a protein structure similar to calcium dependent lectins. Reg I and II have been implicated in control of pancreatic development and may play a role in maintenance of the beta-cell mass in the mature pancreas. Administration of reg I protein has been used in experimental animals as a therapy for surgically-induced diabetes mellitus. Reg I protein is also an inhibitor of calcium carbonate crystalization, important in maintaining the fluidity of pancreatic juice. The reg III gene, whose protein product is pancreatitis associated protein, is induced during pancreatic inflammation. Serum levels of reg III protein are a sensitive marker of severity of pancreatitis. It is an endogenous pancreatic factor that prevents the bacteria infection and scavenges oxygen-derived free radicals. Reg mRNA has been detected in non-pancreatic tissue such as the enterochromaffin-like cells of the stomach, neoplastic tissues of the colon, the small intestine, nervous system, liver tumors, and pituitary. Reg proteins are mitogens to intestinal epithelial cells, pancreatic ductal, beta cells, and Schwann cells, and are likely important to the overall integrity of the pancreas and gastrointestinal tract. 相似文献
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Identification of a novel Reg family gene, Reg IIIdelta, and mapping of all three types of Reg family gene in a 75 kilobase mouse genomic region 总被引:3,自引:0,他引:3
Abe M Nata K Akiyama T Shervani NJ Kobayashi S Tomioka-Kumagai T Ito S Takasawa S Okamoto H 《Gene》2000,246(1-2):111-122
Regenerating gene (Reg), first isolated from a regenerating islet cDNA library, encodes a secretory protein with a growth stimulating effect on pancreatic beta cells that ameliorates the diabetes of 90% depancreatized rats and non-obese diabetic mice. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of three subtypes (types I, II, III) based on the primary structures of the encoded proteins of the genes. We have isolated three types of mouse Reg family gene (Reg I, Reg II, Reg IIIalpha, Reg IIIbeta and Reg IIIgamma) [Unno et al. (1993) J. Biol. Chem. 268, 15974-15982; Narushima et al. (1997) Gene 185, 159-168]. In the present study, by Southern blot analysis of a mouse bacterial artificial chromosome clone containing the five Reg family genes in combination with PCR cloning of every interspace fragment between adjacent genes, the Reg family genes were mapped to a contiguous 75kb region of the mouse genome according to the following order: 5'-Reg IIIbeta-Reg IIIalpha-Reg II-Reg I-Reg IIIgamma-3'. In the process of ordering the genes, we sequenced the 6.8kb interspace fragment between Reg IIIbeta and Reg IIIalpha and encountered a novel type III Reg gene, Reg IIIdelta. This gene is divided into six exons spanning about 3kb, and encodes a 175 amino acid protein with 40-52% identity with the other five mouse Reg (regenerating gene product) proteins. Reg IIIdelta was expressed predominantly in exocrine pancreas, but not in normal islets, hyperplastic islets, intestine or colon, whereas both Reg I and Reg II were expressed in hyperplastic islets and Reg IIIalpha, Reg IIIbeta and Reg IIIgamma were expressed strongly in the intestinal tract. Possible roles of Reg IIIdelta and the widespread occurrence of the Reg IIIdelta gene in mammalian genomes are discussed. 相似文献
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The regenerating (Reg) family comprises an extensive, diversified group of proteins with homology to C-type lectins. Several members of this family are highly expressed in the gastrointestinal tract under normal conditions, and often show increased expression in inflammatory bowel disease. However, little is known about Reg protein function, and the carbohydrate ligands for these proteins are poorly characterized. We report here the first expression and purification of Reg proteins using a bacterial system. Mouse RegIIIgamma and its human counterpart, HIP/PAP, were expressed in Escherichia coli, resulting in the accumulation of aggregated recombinant protein. Both proteins were renatured by arginine-assisted procedures and were further purified using cation-exchange chromatography. The identities of the purified proteins were confirmed by SDS-PAGE, N-terminal sequencing, and MALDI-TOF mass spectrometry. Size exclusion chromatography revealed that both proteins exist as monomers, and circular dichroism showed that their secondary structures exhibit a predominance of beta-strands which is typical of C-type lectins. Finally, both RegIIIgamma and human HIP/PAP bind to mannan but not to monomeric mannose, giving initial insights into their carbohydrate ligands. These studies thus provide an essential foundation for further analyses of human and mouse RegIII protein function. 相似文献
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目的:探讨人再生基因Ⅳ(REGIV)在前列腺细胞中的表达及意义.方法:构建REGIV基因的全序列过表达质粒.将全序列过表达质粒采用脂质体转染的方式转入前列腺细胞系PC-3中.应用Real-time-PCR方法检测REGIV基因mRNA表达,Westembloting检测REGIV基因蛋白质表达,MTT法分析细胞增殖活性.结果:通过荧光显微镜观察计数,细胞转染成功.REGIV基因的全序列过表达使REGIV基因mRNA的表达,蛋白表达提高,细胞增殖能力增强.结论:应用全序列过表达技术可以使前列腺癌REGIV表达水平特异性增高.前列腺癌增值能力的增强说明REGW可能与肿瘤快速增长有关. 相似文献
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Molecular cloning, expression and chromosomal localization of a novel human REG family gene, REG III
Nata K Liu Y Xu L Ikeda T Akiyama T Noguchi N Kawaguchi S Yamauchi A Takahashi I Shervani NJ Onogawa T Takasawa S Okamoto H 《Gene》2004,340(1):161-170
Regenerating gene (Reg), first isolated from a regenerating islet cDNA library, encodes a secretory protein with a growth stimulating effect on pancreatic beta cells that ameliorates the diabetes of 90% depancreatized rats and non-obese diabetic mice. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of four subtypes (types I, II, III, IV) based on the primary structures of the encoded proteins of the genes [Diabetes 51(Suppl. 3) (2002) S462]. Plural type III Reg genes were found in mouse and rat. On the other hand, only one type III REG gene, HIP/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis-associated protein), was found in human. In the present study, we found a novel human type III REG gene, REG III. This gene is divided into six exons spanning about 3 kilobase pairs (kb), and encodes a 175 amino acid (aa) protein with 85% homology with HIP/PAP. REG III was expressed predominantly in pancreas and testis, but not in small intestine, whereas HIP/PAP was expressed strongly in pancreas and small intestine. IL-6 responsive elements existed in the 5'-upstream region of the human REG III gene indicating that the human REG III gene might be induced during acute pancreatitis. All the human REG family genes identified so far (REG Ialpha, REG Ibeta, HIP/PAP, REG III and REG IV) have a common gene structure with 6 exons and 5 introns, and encode homologous 158-175-aa secretory proteins. By database searching and PCR analysis using a yeast artificial chromosome clone, the human REG family genes on chromosome 2, except for REG IV on chromosome 1, were mapped to a contiguous 140 kb region of the human chromosome 2p12. The gene order from centromere to telomere was 5' HIP/PAP 3'-5' RS 3'-3' REG Ialpha 5'-5' REG Ibeta 3'-3' REG III 5'. These results suggest that the human REG gene family is constituted from an ancestor gene by gene duplication and forms a gene cluster on the region. 相似文献
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Namikawa K Fukushima M Murakami K Suzuki A Takasawa S Okamoto H Kiyama H 《Biochemical and biophysical research communications》2005,332(1):126-134
In this study, we examined the expression of mRNAs for Regenerating gene (Reg)/pancreatitis-associated protein (PAP) family members following hypoglossal nerve injury in rats. In addition to four rat family members (RegI, Reg-2/PAP I, PAP II, and PAP III) that had been identified, we newly cloned and sequenced a type-IV Reg gene in rats. Among these five family members, the expression of Reg-2/PAP I mRNA was predominantly enhanced in injured motor neurons after axotomy. Furthermore, a marked induction of PAP III mRNA was observed in the distal part of the injured nerve. A polyclonal antibody was raised against PAP III, and a Western blotting analysis using this antibody confirmed an increased level of PAP III protein in the injured nerve. These results suggest that Reg family members would be new mediators among injured neurons and glial cells, and may play pivotal roles during nerve regeneration. 相似文献
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Identification of a receptor for reg (regenerating gene) protein, a pancreatic beta-cell regeneration factor 总被引:7,自引:0,他引:7
Kobayashi S Akiyama T Nata K Abe M Tajima M Shervani NJ Unno M Matsuno S Sasaki H Takasawa S Okamoto H 《The Journal of biological chemistry》2000,275(15):10723-10726
Reg (regenerating gene) was isolated as a gene specifically expressed in regenerating islets (Terazono, K., Yamamoto, H., Takasawa, S., Shiga, K., Yonemura, Y., Tochino, Y., and Okamoto, H. (1988) J. Biol. Chem. 263, 2111-2114). Rat and human Reg gene products, Reg/REG proteins, have been demonstrated to stimulate islet beta-cell growth in vitro and in vivo and to ameliorate experimental diabetes. In the present study, we isolated a cDNA for the Reg protein receptor from a rat islet cDNA library. The cDNA encoded a cell surface 919-amino acid protein, and the cells into which the cDNA had been introduced bound Reg protein with high affinity. When the cDNA was introduced into RINm5F cells, a pancreatic beta-cell line that shows Reg-dependent growth, the transformants exhibited significant increases in the incorporation of 5'-bromo-2'-deoxyuridine as well as in the cell numbers in response to Reg protein. A homology search revealed that the cDNA is a homologue to a human multiple exostoses-like gene, the function of which has hitherto been unknown. These results strongly suggest that the receptor is encoded by the exostoses-like gene and mediates a growth signal of Reg protein for beta-cell regeneration. 相似文献
14.
RegIII作为胰岛再生源蛋白(regenerating islet—derived protein,Reg)家族的重要成员,在维持肠道功能方面发挥着重要的作用,本综述通过对RegIII在肠道内表达、抗炎、抗菌和对先天免疫影响方面进行概括总结,为进一步研究提供理论基础。 相似文献
15.
Meng-Ru Ho Yuan-Chao Lou Shu-Yi Wei Wen-Chang Lin Chinpan Chen 《Journal of molecular biology》2010,402(4):682-695
Human RegIV protein, which contains a sequence motif homologous to calcium-dependent (C-type) lectin-like domain, is highly expressed in mucosa cells of the gastrointestinal tract during pathogen infection and carcinogenesis and may be applied in both diagnosis and treatment of gastric and colon cancers. Here, we provide evidence that, unlike other C-type lectins, human RegIV binds to polysaccharides, mannan, and heparin in the absence of calcium. To elucidate the structural basis for carbohydrate recognition by NMR, we generated the mutant with Pro91 replaced by Ser (hRegIV-P91S) and showed that the structural property and carbohydrate binding ability of hRegIV-P91S are almost identical with those of wild-type protein. The solution structure of hRegIV-P91S was determined, showing that it adopts a typical fold of C-type lectin. Based on the chemical shift perturbations of amide resonances, two calcium-independent mannan-binding sites were proposed. One site is similar to the calcium-independent sugar-binding site on human RegIII and Langerin. Interestingly, the other site is adjacent to the conserved calcium-dependent site at position Ca-2 of typical C-type lectins. Moreover, model-free analysis of 15N relaxation parameters and simplified Carr-Purcell-Meiboom-Gill relaxation dispersion experiments showed that a slow microsecond-to-millisecond time-scale backbone motion is involved in mannan binding by this site, suggesting a potential role for specific carbohydrate recognition. Our findings shed light on the sugar-binding mode of Reg family proteins, and we postulate that Reg family proteins evolved to bind sugar without calcium to keep the carbohydrate recognition activity under low-pH environments in the gastrointestinal tract. 相似文献
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Jingshu Wang Xiaoping Zhou Li Ma Yukio Koizumi Toshihiro Sugiyama 《Biochemical and biophysical research communications》2009,380(3):472-477
Regenerating gene (Reg) I has been identified as a regenerative/proliferative factor for pancreatic islet cells. We examined Reg I expression in the regenerating liver of a rat model that had been administered 2-acetylaminofluorene and treated with 70% partial hepatectomy (2-AAF/PH model), where hepatocyte and cholangiocyte proliferation was suppressed and the hepatic stem cells and/or hepatic progenitor cells were activated. In a detailed time course study of activation of hepatic stem cells in the 2-AAF/PH model, utilizing immunofluorescence staining with antibodies of Reg I and other cell-type-specific markers, we found that Reg I-expressing cells are present in the bile ductules and increased during regeneration. Reg I-expressing cells were colocalized with CK19, OV6, and AFP. These results demonstrate that Reg I is significantly upregulated in the liver of the 2-AAF/PH rat model, accompanied by the formation of bile ductules during liver regeneration. 相似文献
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Peter J. Fuller Dianne J. Beveridge Russell G. Taylor 《Journal of cellular biochemistry》1995,58(2):260-267
The process of self-renewal which occurs in the gastrointestinal epithelium is greatly amplified and accelerated during the intestinal adaptation which occurs in the residual ileum after massive small bowel resection (MSBR). As with growth and development, these processes must involve the coordinated regulation of many genes. Several families of nuclear proteins are known to be involved in the control of gene expression during development including the POU-domain genes; their expression has not been characterized in the gastrointestinal tract during normal cellular renewal or adaptation, and POU-domain encoding cDNAs were cloned from ileal RNA. Three known genes were cloned: Oct-1, Brn-1 and Tst-1 but no novel members of this gene family were identified. The encoded sequence for rat Oct-1 differs from that previously reported. Oct-1 is relatively ubiquitously expressed with increased expression during both development and adaptation. Minimal expression of Tst-1 was observed. Brn-1 exhibits limited expression in the adult gastrointestinal tract. but may play a role in the fetal gastrointestinal tract. 相似文献