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

OBJECTIVES: 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.

High-level expression of functional recombinant human coagulation factor VII in insect cells.

Recombinant coagulation factor VII (FVII) is used as a potential therapeutic intervention in hemophilia patients who produce antibodies against the coagulation factors. Mammalian cell lines provide low levels of expression, however, the Spodoptera frugiperda Sf9 cell line and baculovirus expression system are powerful systems for high-level expression of recombinant proteins, but due to the lack of endogenous vitamin K-dependent carboxylase, expression of functional FVII using this system is impossible. In the present study, we report a simple but versatile method to overcome the defect for high-level expression of the functional recombinant coagulation FVII in Sf9 cells. This method involves simultaneous expression of both human gamma-carboxylase (hGC) and human FVII genes in the host. It may be possible to express other vitamin K-dependent coagulation factors using this method in the future.

Neutrophil gelatinase-associated lipocalin acts as a protective factor against H(2)O(2) toxicity.

BACKGROUND: Lipocalin 2 (Lcn2, NGAL) is a member of the lipocalin superfamily for which a variety of functions have been reported. However, the precise biological roles of NGAL are not fully known. We have investigated the ability of NGAL to prevent H(2)O(2) toxicity, which is considered to be the classical inducer of oxidative stress caused by ROS generation in an in vitro model. METHODS: NGAL cDNA was isolated from HepG2 cell line and cloned to pcDNA3.1(+) vector. The construct was transfected to CHO cell line. Stable clones were generated, and the expression of NGAL was determined by RT-PCR, Western blot analysis and ELISA. NGAL gene in A549 cell line was downregulated with the siRNA. CHO and A549 cells were intoxicated with H(2)O(2) and cell proliferation was performed by MTT assay. Apoptotic cells were detected by flow cytometry. RESULTS: Cell proliferation was higher in CHO expressing NGAL in doses of 5 and 10 mM H(2)O(2) after 2h compared with the control. H(2)O(2) was also more toxic in the presence of NGAL siRNA compared with the control in A549 cell. Our results also revealed that NGAL protect cells from apoptosis. CONCLUSIONS: Overall, our results revealed for the first time a new function for NGAL/Lcn2: acting as a protective factor against H(2)O(2) toxicity. In the future, NGAL may have the potential application to ameliorate the toxicity induced by oxidative stress conditions.