Isolation, Cloning and High- Level Expression of Neutrophil Gelatinase-Associated Lipocalin Lipocalin2 by Baculovirus Expression System through Gateway Technology

Document Type: Original Article

Authors

1 Blood Transfusion Research Centre, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

2 Department of Anatomy, Faculty of Medicine, Medical University of Hamadan, Hamadan, Iran

3 Department of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran

Abstract

Objective(s)
Lipocalin 2 (Lcn2) is a 25-kDa glycoprotein that has initially been extracted from neutrophil granules. Expression of Lcn2 is induced under various pathophysiological conditions. It is also known as an early marker of kidney and heart injury. High-level expression of recombinant Lcn2 neutrophil gelatinase-associated(NGAL) in insect cells was the aim of this study.
Materials and Methods
Lcn2 gene was isolated from HepG2 cell line. The PCR product was cloned into TOPO vector to construct TOPO-Lcn2. Then Lcn2 was transferred to Gateway adapted Baculovirus DNA by LR recombination reaction. The recombinant Baculovirus DNA was transfected into insect cell line. Expression of recombinant Lcn2 was detected by RT-PCR, ELISA and western blot analysis.
Results
Insertion of Lcn2 into pENTR/D-TOPO vector was confirmed by using PCR. The accuracy of the nucleotides sequence was verified by DNA sequencing. Transfer of the Lcn2 cDNA into the Baculovirus destination vector by LR recombination reaction was confirmed by amplification of a band of about 860 bp length by using forward Lcn2 primer and V5 reverse primer. Next, Lcn2 protein was detected as a prominent band with approximate molecular weight of 30 kDa in SDS-PAGE and western blot analysis. ELISA results revealed high-level expression of Lcn2 by Spodoptera frugiperda (Sf9) cells.
Conclusion
High-level expression of Lcn2 protein in insect cells is promising for future potential applications. Recombinant Lcn2 might be used for producing monoclonal or polyclonal antibodies and as potential therapeutic agent. Large scale expression and purification are next steps that are on the way.
 

Keywords


1.  Devarajan P. Neutrophil gelatinase-associated lipocalin: A promising biomarker for human acute kidney injury. Biomark Med 2010; 16:265-280.

  2. Gwira JA, Wei F, Ishibe S, Ueland JM, Barasch J, Cantley LG. Expression of neutrophil gelatinase-associated lipocalin regulates epithelial morphogenesis in vitro. J Biol Chem 2005; 280:7875-7882.

3. Mori K, Lee HT, Rapoport D, Drexler IR, Foster K, Yang J, et al. Endocytic delivery of lipocalin-siderophoreiron complex rescues the kidney from ischemia-reperfusion injury. J Clin Invest  2005; 115:610–621.

4. Bahmani P, Halabian R, Rouhbakhsh M, Roushandeh AM, Masroori N, Ebrahimi M, et al. Neutrophil Gelatinase-Associated Lipocalin induces the expression of heme oxygenase-1 and superoxide dismutase 1, 2. Cell Stress Chaperones  2010; 15:395-403.

5. Roudkenar MH, Kuwahara Y, Baba T, Roushandeh AM, Ebishima S, et al. Oxidative stress induced lipocalin 2 gene expression: addressing its expression under the harmful conditions. J Radiat Res (Tokyo) 2007; 48:39-44.

6. Roudkenar MH, Halabian R, Roushandeh AM, Nourani MR, Masroori N, Ebrahimi M, et al. Lipocalin 2 regulation by thermal stresses: Protective role of Lcn2/NGAL against cold and heat stresses. Exp Cell Res  2009; 315:3140-3151.

7. Jarmi T, Agarwal A. Heme oxygenase and renal disease. Curr Hypertens Rep 2009; 11:56-62.

8. Zhang H, Xu L, Xiao D, Xie J, Zeng H, Wang Z, et al. Upregulation of neutrophil gelatinase-associated lipocalin in oesophageal squamous cell carcinoma:significant correlation with cell differentiation and tumour invasion. J Clin Pathol  2007; 60:555–561.

9. Holmes MA, Paulsene W, Jide X, Ratledge C, Strong RK. Siderocalin (Lcn 2) Also  Binds Carboxymycobactins, Potentially Defending against Mycobacterial Infections through Iron Sequestration. Structure (London, England: 1993) 2005; 13:29-41.

10. Schmidt-Ott KM, Mori K, Li JY, Kalandadze A, Cohen DJ, Devarajan P, et al. Dual Action of Neutrophil Gelatinase-Associated Lipocalin. J Am Soc Nephrol 2007; 18:407-413.

11.Breustedt DA, Schönfeld DL, Skerra A. Comparative ligand-binding analysis of ten human lipocalins.  Biochim Biophys Acta  2006; 1764:161-173.

12.  Lee HJ, Lee EK, Lee KJ, Hong SW, Yoon Y, Kim JS. Ectopic expression of neutrophil gelatinase-associated lipocalin suppresses the invasion and liver metastasis of colon cancer cells. Int J Cancer  2006; 118:2490-2497.

13.  Tong Z, Wu X, Ovcharenko D, Zhu J, Chen CS, Kehrer JP. Neutrophil gelatinase-associated lipocalin as a survival factor. Biochem J  2005; 391:441-448.

14. Roudkenar MH, Halabian R, Ghasemipour Z, Roushandeh AM, Rouhbakhsh M, Nekogoftar M, et al. Neutrophil gelatinase-associated lipocalin acts as a protective factor against H(2)O(2) toxicity. Arch Med Res  2008; 39:560-566.

15.Bauer M, Eickhoff JC, Gould MN, Mundhenke C, Maass N, Friedl A. Neutrophil gelatinase-associated lipocalin (NGAL) is a predictor of poor prognosis in human primary breast cancer. Breast Cancer Res Treat  2008; 108:389-397.

16. Cho H, Kim JK. Lipocalin2 expressions correlate significantly with tumor differentiation in epithelial ovarian cancer. J Histochem Cytochem  2009; 57:513-521.

17. Hemdahl AL, Gabrielsen A, Zhu C, Eriksson P, Hedin U, Kastrup J, et al. Expression of neutrophil gelatinase-associated lipocalin in atherosclerosis and myocardial infarction. Arterioscler Thromb Vasc Biol  2006; 26:136-142.

18. Katano M, Okamoto K, Arito M, Kawakami Y, Kurokawa MS, Suematsu N, et al. Implication of granulocyte-macrophage colony-stimulating factor induced neutrophil gelatinase-associated lipocalin in pathogenesis of rheumatoid arthritis revealed by proteome analysis. Arthritis Res Ther  2009; 11:R3.

19. Mishra J, Mori K, Ma Q, Kelly C, Barasch J, Devarajan P. Neutrophil Gelatinase-Associated Lipocalin: A Novel Early Urinary Biomarker for Cisplatin Nephrotoxicity. Am J  Nephrol  2004; 24:307-315.

20. Tong Z, Wu X, Ovcharenko D, Zhu J, Chen CS, Kehrer JP. Neutrophil gelatinase-associated lipocalin as a survival factor. Biochem J  2005; 391:441–448.

21.Vemula M, Berthiaume F, Jayaraman A, Yarmush ML. Expression profiling analysis of the metabolic and inflammatory changes following burn injury in rats. Physiol genomics 2004; 18:87-98.

22. Roudkenar MH, Halabian R, Oodi A, Roushandeh AM, Yaghmaei P,Najar MR, et al. Upregulation of neutrophil gelatinase-associated lipocalin, NGAL/Lcn2,in b-Thalassemia patients. Arch Med Res 2008; 39:402-407

23. Masroori N, Halabian R, Mohammadipour M, Roushandeh AM, Rouhbakhsh M, Najafabadi AJ, et al. High-level expression of functional recombinant human coagulation factor VII in insect cells. Biotechnol Lett  2010; 32:803-809.

24.Cha HJ, Dalal NG, Pham MQ, Vakharia VN, Rao G, Bentley WE, et al. Insect larval expression process is optimized by generating fusions with green fluorescent protein. Biotechnol Bioeng 1999; 65:316-324.

25. Miller L.K. Insect baculoviruses: Powerful gene expression vectors. Bioessays  1989; 11:91-95.

26. Gholoobi A, Sankian M, Zarif R, Farshadzadeh Z, Youssefi F, Sadeghian A, et al. Molecular cloning, expression and purification of protein TB10.4 secreted by mycobacterium tuberculosis. Iran J Basic Med Sci  2010; 13:189-193.