|
|
|
| Construction and Expression of Rat Proprotein Convertase 2 Gene |
| WU Shuang1, ZHOU Dong2, ZHANG Juan-hui2, TANG Song-shan1 |
1. Guangdong Provincial Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Guangzhou 510006, China;
2. Department of Neurosurgery, Guangdong General Hospital, Guangzhou 510080, China |
|
|
|
Abstract Objective: Proprotein convertase 2 (PC2) is one major member of the pro-protein convertases that involve in the maturation of neuroendocrinopeptide precursor. To obtain rat pc2 gene for further studying its construction and function relation in rodent tumor model, rat pc2 gene was recombined. Methods: Total RNAs were extracted from the rat pituitary. The pc2 cDNA was cloned and amplified by using RT-PCR technique. The pc2 DNA was inserted into the pCDNA3.1/Zeo vector. The transfection of recombinant vector pCDNA3.1-pc2 or together with its chaperon partner 7b2 gene successfully expressed and activated the pc2 gene in breast adenocarcinoma cell line 4T1. Conclusion: By gene sequencing, transfection, activation, and Western blot analyses, the pc2 gene was confirmed to be inserted and expressed successfully in mammal cells.
|
|
Received: 30 January 2012
Published: 25 August 2012
|
|
|
|
|
[1] Abdel-Majid Khatib, Ge'raldine Siegfried. Proprotein convertases in tumor progression and malignancy. American Journal of Pathology, 2002,160(6):1921-1935.
[2] Seidah N G, Prat A. Precursor convertases in the secretory pathway, cytosol and extracellular milieu. Essays Biochem, 2002, 38:79-94.
[3] Thomas G. Furin at the cutting edge: from protein trafic to embryogenesis and disease. Nat Rev Mol Cell Biol, 2002,3(10):753-766.
[4] Taylor N A, Van De Ven W J, Creemers W J. Curbing activation: proprotein convertases in homeostasis and pathology. FASEB J, 2003,17:1215-1227.
[5] Leak T S, Keene K L, Langefeld C D, et al. Association of the proprotein convertase subtilisin/ kexin-type 2 (PCSK2) gene with type 2 diabetes in an African American population. Mol Genet Metab, 2007,92 (1-2):145-150.
[6] Tian S, Jianhua W.Comparative study of the binding pockets of mammalian proprotein convertases and its implications for the design of specific small molecule inhibitors. International Journal of Biological Sciences, 2010,6(1):89-95.
[7] Seidah N G, Mowla S J, Hamelin J, et al. Mammalian subtilisin/kexin isozyme SKI-1: a widely expressed proprotein convertase with a unique cleavage specificity and cellular localization.Proc Natl Acad Sci USA, 1999, 96(4):1321-1326.
[8] Sakai J, Rawson R B, Espenshade P J, et al. Molecular identification of the sterol-regulated luminal protease that cleaves SREBPs and controls lipid composition of animal cells. Mol Cell, 1998, 2(4):505-514.
[9] Ye J, Rawson R B, Komuro R, et al. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol Cell, 2000, 6:1355-1364.
[10] Shennan K I, Taylor N A, Jermany J L, et al. Differences in pH optima and calcium requirements for maturation of the prohormone convertases PC2 and PC3 indicates different intracellular locations for these events, J Biol Chem, 1995,270(3):1402-1407.
[11] Espinosa V P, Liu Y, Ferrini M, et al. Differential regulation of PC1/3,PC2 and phosphorylated creb by short-team and long-team morphine treatment: implication for understanding the "switch" to opiate addiction. Neuroscience, 2008,156(3): 788-799.
[12] Bl醶quez M, Thiele C, Huttner W B, et al. Involvement of the membrane lipid bilayer in sorting prohormone convertase 2 into the regulated secretory pathway. Biochem J, 2000,349(8):843-852.
[13] Tang S S, Zhang J H, Liu H X, et al. PC2 / CPE-mediated pro-protein processing in tumor cells and its differentiated cells or tissues. Mol Cell Endocrinol, 2009, 303 (1-2): 43-49.
[14] Furuta M, Zhou A, Webb G, et al.Severe defect in proglucagon processing in islet A-cells of prohormone convertase 2 null mice. J Biol Chem, 2001,276 (29):27197-27202.
[15] Benjannet S, Rondeau N, Day R, et al. PC1/3 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci, 1991, 88:3564-3568. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
