Coding sequences for RHO proteins and IQGAP1 variants were amplified using sequence-specific primer and cloned into the expression vector. Different pGEX vectors encoding an N-terminal glutathione S-transferase (GST) fusion protein were used for the overexpression ofvarious human genes: different IQGAP1 (accession number P46940) regions (aa 863–1345, 863–1657, 877–1558, 1276–1657, 1276–1575, 1576–1657) and also C-terminal truncated RAC1 (aa 1–179), CDC42 (aa 1–178), and RHOA (aa 1–181). pMCSG7 vector was used for overexpression of IQGAP1 aa 962–1345 and pET46 EkLIC vector (Merck) for the overexpression of IGQAP1 S1441E and S1143E mutants (aa 877–1558) as His-tagged proteins. The Kazusa cDNA clone KIAA0051 (70 (link)) was used as a template for site-directed mutagenesis.
Pet46 eklic vector
Manufactured by Merck Group
The PET46 EkLIC vector is a laboratory tool used for cloning and protein expression. It is designed for efficient ligation-independent cloning and enables the production of recombinant proteins in Escherichia coli cells. The vector provides a T7 promoter for high-level expression and contains an N-terminal His-tag sequence for convenient protein purification.
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Lab products found in correlation
2 protocols using pet46 eklic vector
1
Overexpression of IQGAP1 and RHO Proteins
2
Purification of Recombinant β-Catenin
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A construct comprising residues 126–686 of human β-catenin (AB451264) was sub-cloned into pET-46 Ek/LIC vector (EMD Millipore). The recombinant His-fusion β-catenin was expressed in E. coli BL21 (DE3) and purified by affinity chromatography using nickel-nitrilotriacetic acid-agarose (HisPur™ Ni-NTA, Thermo Scientific). The protein was eluted from the resin with 250 mM imidazole, 150 mM NaCl, 20 mM Tris, pH 8.0, and purified further on a gel-filtration HiLoad 16/60 Superdex-200 column (GE Healthcare) equilibrated with buffer (150 mM NaCl, 20 mM CAPS, pH 10.5).
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