Ekaterina Heldwein
  • Associate Professor with tenure, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA
Research fields
  • Microbiology
Identification of Buffer Conditions for Optimal Thermostability and Solubility of Herpesviral Protein UL37 Using the Thermofluor Assay
Authors:  Andrea L. Koenigsberg, Jared D. Pitts and Ekaterina E. Heldwein, date: 06/20/2020, view: 2964, Q&A: 0
Structural and biochemical studies of proteins require high amounts of stable, purified proteins. Protein stability often depends on the buffer composition, which includes pH and concentration of salts or other solutes such as glycerol, hence an efficient method for identifying optimal buffer conditions for stability would minimize time and resources used for protein purification and further studies. This protocol describes the use of the Thermofluor assay, in combination with a custom 24-condition screen, to identify buffer conditions that increase protein thermostability, using the conserved herpesviral protein UL37 as an example. Detailed instructions on screen conditions, running the Thermofluor MATLAB script, and analyzing the data are provided. In comparison to circular dichroism (CD), the buffer screen in combination with Thermofluor assay provides a faster and more informative method to analyze protein thermostability.
Expression, Purification and Crystallization of the Herpesvirus Nuclear Egress Complex (NEC)
Authors:  Janna M. Bigalke and Ekaterina E. Heldwein, date: 07/20/2016, view: 7801, Q&A: 0
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.
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