Abstract
The protocol describes the production and crystallization of the soluble form of the nuclear egress complex (NEC) from Herpes simplex virus 1 and Pseudorabies virus. The NEC is a heterodimer that consists of conserved proteins UL31 and UL34. NEC oligomerization deforms the inner nuclear membrane around the capsid in infected cells, thereby mediating capsid budding into the perinuclear space during nuclear egress. We have successfully developed a protocol for large-scale preparation of highly pure NEC from two different viruses in a prokaryotic expression system, which enabled us to crystallize these viral protein complexes and determine their structures. This procedure may be adapted to purify and crystallize other soluble protein complexes.
Keywords: Nuclear egress complex, Protein expression in E. coli, Protein purification, Crystallization
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. 12% SDS-PAGE analysis of pure HSV-1 and PRV NEC Figure 2. Crystals of HSV-1 NEC (A) and PRV NEC (B). Crystals were grown in hanging drops at 22 °C. The reservoir solution for growing HSV-1 NEC crystals contained 10% PEG 8000, 0.1 M Na citrate, pH 5.6, 5 mM NiCl2, and for PRV NEC crystals 18% PEG 3350, 0.3 M NaSCN, 0.3 M NaCl.
Notes
Crystallization of HSV-1 NEC depends on the formation of a disulfide bond involving Cys278UL31. Therefore, not more than 0.5 mM TCEP may be used during purification. Both protein complexes should be purified and frozen within 40 h to prevent degradation and aggregation.
Recipes
Acknowledgments
This work was funded by the NIH grants 1R21AI097573 and 1R01GM111795 (E.E.H.), the Burroughs Wellcome Fund (E.E.H.), and by the postdoctoral fellowship from the Deutsche Forschungsgemeinschaft GZ: BI 1658/1-1 (J.M.B.). Parts of this protocol were published previously in Bigalke et al., 2014 and Bigalke & Heldwein, 2015.
References
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