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L. Cheng, N. Perdue, Q. Yan; A Recombinant Flag–Tagged SPARC Fusion Protein Expressed in Baculovirus System . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2567.
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© ARVO (1962-2015); The Authors (2016-present)
SPARC is a secreted glycoprotein that plays an important role in the maintenance of lens transparency. To study the molecular mechanisms of how SPARC regulates lens cell activities and interacts with other proteins, significant amounts of pure, functional protein are required for experiments in vitro. The aim of this study is to establish an expression system for producing sufficient quantities of biologically active flag–tagged murine (m) SPARC recombinant protein. FLAG epitope enables straightforward purification of the protein and will be used for detection of SPARC binding proteins.
Mouse SPARC cDNA, minus the signal sequence, was amplified by PCR using cDNA of mouse lens epithelial cells. The primer sequences were flanked with attB1 and attB2 to create the recombination sites. The amplified fragment was cloned into pDONRTM221 vector, and subcloned into the expression vector pFBIF which was derived from pFastBac1. pFBIF contains a fragment encoding a signal peptide of human immunoglobulin, and a FLAG peptide. Bacmid DNA was isolated from the DH10BAC competent cells that were transformed with the pFBIF containing the mSPARC cDNA. Bacmid DNA was transfected into the Spodoptera frugiperda (Sf21) cells to yield FLAG–mSPARC fusion protein. The secreted FLAG–mSPARC protein was purified using anti–FLAG M1 Agarose Affinity Gel. The purified recombinant protein was evaluated by SDS–PAGE and Western blot analyses; its biological activity was assayed by inhibition of 3H–thymidine incorporation of lens epithelial cells.
The recombinant protein was expressed in (Sf21) insect cells. The highest level of expression was achieved after 4 days of incubation. The yield of purified FLAG–tagged mSPARC was up to 20mg/L culture. The purity of the fusion protein was greater than 95% by SDS–PAGE. Western blot analysis of the recombinant fusion protein with antibody against mSPARC or FLAG produced a band of approximately 43 kDa under reducing condition, and 37 kDa under non–reducing condition. FLAG–mSPARC showed inhibition of lens epithelial cell proliferation in the presence of 2% fetal bovine serum in culture medium.
We established an efficient method for expression and purification of biologically active FLAG–tagged mSPARC protein in insect cells. Production of significant amounts of FLAG–tagged SPARC protein opened up the opportunity to investigate biological functions of SPARC in lens epithelial cells and to identify SPARC binding partners.
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