Protein expression for structural studies
Microbial Cell Factories volume 5, Article number: P40 (2006)
Protein structure determination is an essential tool for studying protein function, and assists the design of novel drugs. The goal of the Israel Structural Proteomics Center (ISPC) is to determine the structures of proteins related to human health in their functional context http://www.weizmann.ac.il/ISPC. One of the bottlenecks encountered, primarily for eukaryotic proteins, is the production of soluble and correctly folded proteins suitable for crystallization trials. In order to overcome this obstacle, we have applied various expression strategies. E. coli is the expression system of choice, in which different parameters are tested in parallel. In cases in which post-translational modification is essential for obtaining a functional protein, eukaryotic expression systems such as Pichia pastoris or baculovirus are being employed.
Each target is expressed in parallel in several expression vectors in E. coli. Design of vectors, which harbor the same restriction sites and code for a cleavable N-terminal His-tag facilitate DNA cloning and purification, respectively. When necessary, a target protein is co-expressed with its natural binding partners or with molecular chaperones. Results of a representative protein expression experiment are shown below. Figure 1 demonstrates the effect of co-expression of molecular chaperones in E. coli on protein solubility. Levels of soluble protein were significantly increased when the protein was co-expressed with certain combinations of molecular chaperones (Figure 1, C and 1D).
Rapid evaluation of protein expression under small-scale culture conditions is essential for implementing an efficient high-throughput protein production process. Parallel utilization of a repertoire of protein expression strategies resulted in a significant increase in the number of soluble proteins obtained.
Albeck S, Burstein Y, Dym O, Jacobovitch Y, Levi N, Meged R, Michael Y, Peleg Y, Prilusky J, Schreiber G, Silman I, Unger T, Sussman JL: Three-dimensional structure determination of proteins related to human health in their functional context at the Israel Structural Proteomics Center (ISPC). Acta Crystallogr D Biol Crystallogr. 2005, 61: 1364-72. 10.1107/S0907444905023565.
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Peleg, Y., Albeck, S., Burstein, Y. et al. Protein expression for structural studies. Microb Cell Fact 5 (Suppl 1), P40 (2006). https://doi.org/10.1186/1475-2859-5-S1-P40