Expression and purification of recombinant human Cosmc extracellular domain in Sf-9 insect cells / 中国免疫学杂志

Objective:In the present study,Bac-to-Bac baculovirus expression system was used to obtain recombinant human Cosmc extracellular domain protein,which can lay the foundation for the research about the structure and function of Cosmc protein in vitro,and simultaneously provide ideas for the research of O-glycosylation and related diseases. Methods: The Cosmc extracellular domain ( Cosmc-ED) gene was cloned into a transfer vector pFastBac1 to form the recombinant donor plasmid pFastBac1-Cosmc ED, which was transformed into competent cells DH10Bac. By using blue-white selection and PCR analysis,we could obtain recombinant shuttle vector rBacmid-Cosmc ED. Then, the recombinant gene DNA of rBacmid-Cosmc ED was used to transfect Sf-9 mediated by cationic lipid formulation,and the recombinant baculovirus bacmid was obtained,which was further used to infect the serum-free cell Sf-9 to express Cosmc-ED in the supernatant. Then the protein of interest was detected by SDS-PAGE and Western blot and purified with Ni-NTA affinity column. Results:SDS-PAGE and Western blot analysis showed a specific band about 33 kD,consistent with the interest protein. Mass spectrometry results further prove that the protein was Cosmc extracellular domain protein. Conclusion: Human Cosmc-ED protein can be successfully expressed in Sf-9 insect cells and laid basis for subsequent studies.

Protein Expression of the Bombyx mori Decapentaplegic Gene using the Baculovirus Expression Vector System

The Bombyx mori decapentaplegic gene is one of the conserved genes in vertebrate and invertebrates. The TGF-beta superfamily contains conserved polypeptide growth factors that play important roles in different cellular processes such as proliferation, apoptosis, differentiation and cell-fate determination. The B. mori dpp gene shares genetic homology with hBMPs and Drosophila dpp. Until now, only few studies have been conducted to examine the functions of B. mori dpp; and hence, its function is not yet well understood. In this study, the baculovirus expression vector system (BEVS) was used for expression of the recombinant B. mori dpp protein and in which the recombinant baculovirus is recovered in the host Sf9 cells. The selected pure recombinant baculovirus containing B. mori dpp gene (rBV-egfp-Bm dpp) was used to increase the effective protein purification by using His-tag extraction strategy. After selection of recombinant baculovirus, recombinant B. mori dpp proteins were extracted from the re-infected cells with pure rBV-egfp-Bm dpp. Herein, we summarize the efficient expression and purification of B. mori dpp proteins from the insect cells using the BEVS. This recombinant protein could be suitable for functional test and various application studies.