The objective is to be able to produce NMR """"""""structurable"""""""" samples for the NESG while at the same time developing methods for the efficient production of eukaryotic proteins and domains for NMR. The targets that we are going to pursue are subsets of NESG's main target group. Cancer-related targets and those involved in human ubiquitylation pathways will be the main focus, along with community- and PSI-Biology Center-nominated targets. We will use bioinformatics and multiple domain boundaries as our first approach to identify which protein constructs will be most amenable to NMR analysis. Experimental domain mapping via limited proteolysis followed by mass spectrometry will be used as a second approach. The constructs will be cloned into one of two pET vectors using standard PCR techniques and Ligation independent cloning (LIC), in a 96-well format. All the proteins will be expressed in E.coli grown in 15N-labelled minimal media and purified using batch Ni-affinity purification methods. All targets that are deemed amenable to structure determination by NMR will be labeled with 13C and 15N and more extensively purified and assessed for stability. For the more difficult eukaryotic targets, new methods will be developed to improve the efficiency with which we can purify soluble, stable domains. A large proportion of our efforts will devoted to implementing the bacullovirus expression system in a high throughput manner for selected human targets. Any proteins resulting from this endeavor will be sent to other NESG laboratories for crystallization trials.
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