a, Schematic of the self-cleaving polyprotein construct used to express recombinant influenza B heterotrimeric polymerase in insect cells. N-terminally it encodes the tobacco etch virus (TEV) protease that cleaves C-terminal to the amino-acid sequence ENLYFQ (in italics) and releases N-terminally His-tagged PA, PB1, C-terminally Strep-tagged PB2 and cyan fluorescent protein (CFP) for facilitated expression monitoring. Arrows indicate the N-to-C-terminal direction and the termini of each mature protein. The histidine and streptavidin tags are underlined. b, Using the PB2 C-terminal strep-tag, most contaminating proteins could be separated from the polymerase as judged by 10% SDS–PAGE followed by Coomassie blue staining. Lanes ‘M’ contain the protein markers (molecular masses indicated); ‘in’, ‘ft’ and ‘w’ denote the input, flow-through and wash of the engineered streptavidin (strep-tactin) column, respectively, and ‘elution’ indicates the re-mobilization of bound heterotrimeric polymerase by a sharp gradient of d-desthiobiotin. The three subunits, PA (85.7 kDa), PB1 (86.1 kDa) and PB2 (90.8 kDa), run together on the gel. c, After ammonium sulphate precipitation, IMAC, strep-tactin affinity and heparin chromatography, the final purification step consists of size-exclusion chromatography. The elution profile (monitored by the absorbance at 280 nm) with a single and nearly symmetric peak suggests a homogeneous and monomeric polymerase complex. d, Recombinant influenza B polymerase was analysed by electron microscopy following negative staining with sodium silico-tungstate of 0.02 mg ml −1 protein sample. The image demonstrates that the sample is homogeneous and monodisperse with a V- or doughnut-like shape with a central cavity.