title: Expression and Purification of Class 7 Semaphorin and Its PlexinC1 Receptor Using Baculovirus-Mediated Mammalian Cell Gene Transduction.
abstract: Semaphorins and their receptor plexins are large glycoproteins that are difficult to express using regular recombinant methods, and the widely used E. coli and baculovirus-insect cell systems have been inadequate for semaphorins and plexins which contain a large number of domains and are heavily modified by glycosylation. Here, we describe the expression of class 7 semaphorin (Sema7A) and the extracellular domain of its receptors PlexinC1, using the baculovirus-mediated mammalian cell gene transduction (BacMam) method. A robust mammalian cell expression gene cassette, including a highly efficient secretion signal peptide, is introduced into the baculovirus which subsequently enters mammalian cells for efficient expression in suspension cell culture. Large amount of high-infectivity BacMam viruses are needed for infecting suspended mammalian cells in large scale, to generate semaphorin and plexin proteins at an amount sufficient for binding experiments and crystallographic studies. The inclusion of serum in expression ensures the robustness of cell culture, but introduces substantial amount of contaminant proteins interfering with immobilized metal ion affinity purification, which can be overcome with a two-step purification scheme.
authors: Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Morten 7-615, 303 E Chicago Avenue, Chicago, IL, 60611, USA.
authors: Xiaoyan
authors: X
authors: Chen
authors: Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Morten 7-615, 303 E Chicago Avenue, Chicago, IL, 60611, USA.
authors: Po-Han
authors: PH
authors: Chen
authors: Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Morten 7-615, 303 E Chicago Avenue, Chicago, IL, 60611, USA. x-he@northwestern.edu.
authors: Xiaolin
authors: X
authors: He
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002850:Chromatography, Gel; D057809:HEK293 Cells; D006801:Humans; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011991:Receptors, Virus / Q000302:isolation & purification / Q000378:metabolism*; D039961:Semaphorins / Q000235:genetics* / Q000302:isolation & purification; D061987:Sf9 Cells; D014161:Transduction, Genetic
chemical_list: D011956:Receptors, Cell Surface; D011991:Receptors, Virus; D039961:Semaphorins; C112038:VESPR semaphorin receptor
keywords: BacMam; Baculovirus; Cell-surface receptor; Glycoprotein; Mammalian cell expression; Plexin; Semaphorin; Suspension mammalian cell culture