The staff of the Protein Puriication Core (PPC) use a number of techniques for effective protein production. The PPC has access to a wide variety of tools for the expression of recombinant protein in Escherichia coli, including many types of plasmid expression vectors and specialized bacterial strains. There is a rather large collection of strains to choose from, with genetic defects that influence proteolytic activity, mRNA stability, membrane permeability, and intracellular redox potential. In addition, there are strains that overproduce protein disulfide isomerase, molecular chaperones, and redox enzymes for coexpression with target proteins. There is an equally large and diverse collection of bacterial plasmid vectors for recombinant protein expression. Many of these use the Gateway cloning technology, making them quick and easy to use. The PPC staff has experience with all of the major regulatory systems (e.g., T7, tac, PBAD, trc, λPL) and various formats for the production of recombinant proteins (untagged or fused to MBP, GST, NusA, thioredoxin, His-tag, Arg-tag, FLAG-tag and the biotin acceptor peptide) to make full use of these reagents. The PPC personnel are experienced with all standard chromatography techniques required for protein purification. The core maintains a full array of supplies necessary for ion exchange, hydrophobic interaction, lectin, hydroxyapatite, dye, size exclusion, and affinity chromatography. Materials for chromatofocusing are also on hand. In addition to purification technology, the staff is very knowledgeable of methods required to characterize recombinant protein products. Among those used are gel electrophoresis and isoelectric focusing, mass spectroscopy, western analysis, N-terminal sequencing, dynamic light scattering and analytical ultracentrifugation, and circular dichroism spectroscopy. For structural studies, the PPC has in place standard operating procedures for the production of isotopically enriched proteins for heteronuclear Nuclear Magnetic Resonance experiments and selenomethionine-substituted proteins for crystallography. Methods have been established that eliminate the need to change bacterial cell type by manipulating the medium formulation and induction parameters, and produce recombinant protein at levels equivalent to the wildtype expression. For those proteins that fail to crystallize, the core can perform limited proteolysis as a way to identify potential structural domains, providing the Macromolecular Crystallography Laboratory investigator additional avenues for structural studies. This method has been extensively used both analytically, and on a preparative scale to produce structural domains that can be purified using conventional chromatography. The core produces and maintains three different kinds of tobacco etch virus (TEV) protease that are used by the Macromolecular Crystallography Laboratory for in vitro cleavage of fusion proteins that contain an intervening protease recognition sequence. Available are an N-terminal tagged His6-TEV protease, an untagged TEV protease and a Maltose Binding Protein-TEV protease fusion protein. All contain a mutation that minimizes autoinactivation. Each has its advantage depending on the design of the protein purification scheme.
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