Baculovirus‐driven expression and purification of glycine receptor α1 homo‐oligomers

Abstract

The glycine receptor is a figand-gated anion channel protein of postsynaptic membranes. We expressed a homo-oligomeric receptor composed of human al subunits in Spodoptera frugiperda cells by infection with a recombinant Autographa californica nuclear polyhedrosis virus. A substantial fraction of the recombinant receptor was incorporated as a functional channel protein into the cell's plasma membrane at expression levels 4-to 30-fold higher than in other eukaryotic heterologous expression systems or native rat spinal cord membranes, respectively. Upon detergent solubilization, the al receptor was found to exist in a predominantly monodisperse state and could be affinitypurified to near homogeneity. This preparation is a potential starting point for future crystallisation studies.Key words: Glycine receptor; Baculovirus; Affinity purification; Heterologous expression transmembrane topology characterized by an extended N-terminal extracellular domain followed by four transmembrane segments (M1-M4; see [1]).Here, we expressed al homo-oligomeric GlyR in Spodoptera frugiperda (Sf9) insect cells infected with a recombinant Autographa californica baculovirus, in which the GlyR al cDNA was placed under the control of the polyhedrin promoter. The baculovirus Sf9 cell system was previously used to overexpress the c~l GlyR [7] in order to produce preparative amounts of GlyR protein. Receptor purification described in that study involved solubilisation with digitonin/desoxycholate and resulted in a partially proteolysed protein. Here we used solubilisation of the receptor with the more economical conventional detergent Triton X-100 and affinity-purified it to a nearly homogenous and monodisperse state, which appears suitable for crystallization studies.

Oligomerization of Baculovirus LEF-11 Is Involved in Viral DNA Replication

“…Homo-oligomers are often the active forms of many DNA replication-specific expression factors in baculovirus [ 18 , 19 ]. Previous studies have shown that oligomers of several genes, including lef-1 , lef-2 , ie-1 , ie-2 , lef-5 , dbp , ha44 and cg30 , are required for regulating DNA replication during AcMNPV proliferation [ 20 – 26 ].…”

Insertional Mutation of the Drosophila Nuclear Lamin Dm0 Gene Results in Defective Nuclear Envelopes, Clustering of Nuclear Pore Complexes, and Accumulation of Annulate Lamellae

“…After blocking in 10% (wt/vol) nonfat dry milk in PBS overnight at 4°C, the blot was incubated with hybridoma supernatants of the monoclonal Drosophila lamin Dm 0 antibodies U25 (undiluted; kindly provided by H. Saumweber, Humboldt University, Berlin, Germany; Risau et al, 1981 ) and ADL67 (dilution 1:20; kindly provided by N. Stuurman, Biozentrum, Basel, Switzerland; Riemer et al, 1995 ) or of the Drosophila lamin C antibody LC28 (dilution 1:20; also provided by N. Stuurmann; Riemer et al, 1995 ) for 2 h at room temperature. Subsequent incubation with alkaline phosphatase-conjugated anti–mouse antibodies ( Promega Biotech) and visualization with NBT/BCIP were performed as described ( Morr et al, 1995 ).…”