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.

Construction of mcpr1gene vector and expression of mcpr1 in escherichia coli / 实用口腔医学杂志

Objective: To construct mcpr1prokaryoti c expression vector and to express MCPR1 protein.Methods:PCR was used to obtain coding region of mcpr1. Construction of a high-level fusion protein expression vector pGEX-4T-mcpr1 was conducted by inserting the fra gment of coding region of mcpr1into a fusion protein expression vector pGEX -4T-1. Then the recombinant plasmid was transferred into E. colito prepar e the MCPR1/GST fusion protein. DNA sequencing and endonucleases digesting were used to check the coding region. Results:pGEX-4T- mcpr1 wa s constructed successfully and the coding region was inserted into the vector co rrectly. A new protein band of 36 000 was observed by SDS-PAGE analysis after i nduction by IPTG. The 36 000 protein amounted to 39 percent of the total prote in and existed mostly in precipitation of broken bacteria. Conclusion: MCPR1 protein can be expressed in E. coliexpression system and purif ied initially.