Recombinant protein expression and purification.

All constructs were expressed in Escherichia coli Rosetta (Novagen) cells using overnight induction at an optical density at 600 nm (OD₆₀₀) of 0.6 to 1.0 with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 16°C. Cell pellets were resuspended in buffer A [50 mM HEPES, 100 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP)] supplemented with 1 mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride (PMSF), 1× cOmplete protease inhibitor cocktail (Roche), and egg white avidin (Sigma)] and were lysed using either sonication, a French press, or a Microfluidizer. UL21C was captured from the clarified lysate using Strep-Tactin Sepharose resin (GE Healthcare) and was eluted with 5 mM d-desthiobiotin (Sigma) in buffer A.

Prior to crystallization, UL21C was separated from copurifying E. coli nucleic acids (NAs) by use of a HiTrap Heparin Sepharose column (GE Healthcare) in buffer A. UL21C was eluted from heparin in buffer A using a NaCl gradient from 0.1 to 1.0 M. Constructs containing the PreScission protease site were cleaved with recombinant glutathione S-transferase (GST)-tagged HRV 3C (PreScission) protease at a protein/protease mass ratio of 10:1 at 4°C overnight without continued stirring or rocking. After cleavage, the mixture was passed over Strep-Tactin Sepharose and glutathione Sepharose (GE Healthcare) to remove the uncleaved protein, cleaved Strep-tag II, and PreScission protease, and the unbound fraction containing untagged UL21C was collected. Constructs were further purified using size exclusion chromatography (Superdex 200, in buffer A), and were stored with 1× Halt protease inhibitor cocktail (Pierce).

Selenomethionine-containing UL21C was expressed by growing transformed Rosetta cells in M9 minimal medium (1× M9 salts, 0.4% glucose, 2 mM magnesium sulfate, 0.1 mM calcium chloride, 0.2 mg/liter thiamine, 1 mg/liter biotin) at 37°C. At an OD₆₀₀ of 0.5, lysine, phenylalanine, threonine (100 mg/liter), isoleucine, leucine, valine (50 mg/liter), and selenomethionine (60 mg/liter) were added, and the mixture was shaken for 15 min at 37°C. Protein expression was induced with 1 mM IPTG overnight at 20°C. Selenomethionine-containing protein was purified like native protein except that buffer A contained 2 mM TCEP to mitigate selenomethionine oxidation.