Soluble Expression and Purification of the Large Extracellular Loop of the Human TSPAN12 Protein from Escherichia Coli

Abstract

Objective: To construct a prokaryotic expression plasmid for the large extracellular loop (LEL) of human TSPAN12 protein and obtain highly purified soluble recombinant proteins. Methods: In this experimental study, the coding sequence of Tspan12 LEL was first cloned into pMal-c2x to link with the coding sequence of the maltose binding protein (MBP). Then the DNA of MBP-tagged Tspan12 LEL (MBP-TSPAN12 LEL) was cloned to multiple cloning site 1 of vector pETDuet-1 after PCR amplification, restriction of enzyme digestion and T4 ligase reaction. DNA of DsbC was cloned into multiple cloning site 2 of vector pETDuet-1 and co-expressed with MBP-TSPAN12 LEL in order to facilitate disulfide bond formation. After transforming the recombinant plasmid into OrigamiB (DE3), MBP-TSPAN12 LEL was expressed by isopropyl-β-d-thiogalactoside induction and purified by amylose resin affinity chromatography and anion exchange chromatography. Results: Sequencing results suggested that recombinant plasmid was successfully constructed. SDS-PAGE showed that the molecular weight of the soluble MBP-TSPAN12 LEL was about 60 kD. Abundant, soluble and highly purified fusion protein was acquired after affinity and anion exchange chromatography. Conclusions: The experimental results prove that co-expression DsbC with MBP-TSPAN12 LEL is a practicable way to produce soluble large extracellular loop of human Tspan12 protein in Escherichia coli. Key words: TSPAN12; maltose binding protein; prokaryotic expression; soluble protein; purification