Three-dimensional structure determination of proteins related to human health in their functional context at The Israel Structural Proteomics Center (ISPC)

Abstract

The principal goal of the Israel Structural Proteomics Center (ISPC) is to determine the structures of proteins related to human health in their functional context. Emphasis is on the solution of structures of proteins complexed with their natural partner proteins and/or with DNA. To date, the ISPC has solved the structures of 14 proteins, including two protein complexes. It has adopted automated high-throughput (HTP) cloning and expression techniques and is now expressing in Escherichia coli, Pichia pastoris and baculovirus, and in a cellfree E. coli system. Protein expression in E. coli is the primary system of choice in which different parameters are tested in parallel. Much effort is being devoted to development of automated refolding of proteins expressed as inclusion bodies in E. coli. The current procedure utilizes tagged proteins from which the tag can subsequently be removed by TEV protease, thus permitting streamlined purification of a large number of samples. Robotic protein crystallization screens and optimization utilize both the batch method under oil and vapour diffusion. In order to record and organize the data accumulated by the ISPC, a laboratory informationmanagement system (LIMS) has been developed which facilitates data monitoring and analysis. This permits optimization of conditions at all stages of protein production and structure determination. A set of bioinformatics tools, which are implemented in our LIMS, is utilized to analyze each target.

Protein production and purification

“…In both cases, the cells can, and should, be grown to high densities (OD 600 of [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] in highly enriched medium47 in baffled shake flasks48 , 49. Whatever the final cell density, it is advisable to induce the expression of the T7 RNA polymerase at mid-to-late log phase of the growth curve to ensure maximal yield while avoiding the problems associated with cells going into stationary phase (for example, induction of proteases).…”

Automation in biological crystallization

“…Other systems automatically dispense the aqueous solutions directly into the paraffin oil, with small amounts of sample being dispensed first, followed by the cocktail solution. In this case, centrifuging the plates may be needed to coalesce the drops and form the experimental droplets (Luft et al, 2003;Albeck et al, 2005). An advantage of this system is the possibility of accurately dispensing very small volumes (<100 nl) into a liquid (the paraffin oil).…”

“…These efforts have resulted in the establishment of several academic (Heinemann et al, 2000;Watanabe et al, 2002;Luft et al, 2003;Albeck et al, 2005;Mueller-Dieckmann, 2006;Mueller et al, 2012) and industrial (Peat et al, 2002;Hosfield et al, 2003) highthroughput facilities.…”

Structural Genomics and Structural Proteomics: A Global Perspective

“…The Israel Structural Proteomics Center (ISPC) (http://www.weizmann.ac.il/ISPC) was established by scientists from the Weizmann Institute of Science, Rehovot, ISRAEL to increase the efficiency of all stages of 3D protein structure determination. 20 Targets submitted to the ISPC are primarily related to human health and disease. The center has a unique combination of scientific expertise and state-of-the-art instrumentation for high-throughput production and crystallization of proteins.…”