The staff of the Protein Purification 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 bacteria(our primary expression system), 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, transfer RNAs, 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 (Life Technologies), 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, lambda PL) and various formats for the production of recombinant proteins (untagged or fused to MBP, GST, NusA, thioredoxin, His-tag, Arg-tag, FLAG-tag, biotin acceptor peptide, etc.) to make full use of these reagents. The PPC has recently established an insect cell protein production facility to compliment its bacterial production capability, using both the Bac-to-Bac baculovirus expression system (Invitrogen)and the Drosophila expression system (Life Technologies). Like most bacterial production, the insect cell facility utilize Gateway cloning technology to maximize productivity. The PPC plans to evaluate the flashBAC baculovirus expression system from Oxford Expression Technologies. Indications are that this system may be valuable for secretory and membrane-bound protein production, and may be a good complement for the Drosophila expression system. 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 for bacteria that eliminate the need to change 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 His7-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. Similarly, the core produces and maintains two types of tobacco vein mottling virus (TVMV) protease also used for in vitro cleavage of fusion proteins. These are available as an N-terminal His6-tagged TVMV protease and an untagged TVMV protease. The protease recognition site is different from TEV protease and allows the use of both recognition sequences in a single fusion protein. As an alternative to these potyvirus proteases, the PPC has recently purified the human rhinovirus 3C protease (i.e., PreScission protease) using a bacterial expression plasmid obtained from Arie Geerlof (Helmholtz Center Munich, Institute of Structural Biology, Neuherberg, Germany). This protease has good activity even at 4C and will be quite useful in cleaving fusion proteins produced in several commercial vectors such as the pGEX-P series, pTriEx-9 and pET-47b(+). In addition, the PPC has also purified the glycosidases endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F1 using bacterial expression plasmids obtained from Daniel Leahy (Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland). These enzyme will be invaluable at removing asparagine-linked oligosaccharide side chains from glycoproteins produced by our insect cell production facility, which often impede the crystallization process. For FY2013, as part of our research support for the Macromolecular Crystallography Laboratory, the PPC has completed 39 cloning projects and performed 85 protein purification. In addition 99 insect cell protein productions at the pilot and preparative levels were completed.

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Miller Jenkins, Lisa M; Feng, Hanqiao; Durell, Stewart R et al. (2015) Characterization of the p300 Taz2-p53 TAD2 complex and comparison with the p300 Taz2-p53 TAD1 complex. Biochemistry 54:2001-10
Shaw, Gary X; Li, Yue; Shi, Genbin et al. (2014) Structural enzymology and inhibition of the bi-functional folate pathway enzyme HPPK-DHPS from the biowarfare agent Francisella tularensis. FEBS J 281:4123-37
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Lountos, George T; Tropea, Joseph E; Waugh, David S (2011) Structure of human dual-specificity phosphatase 27 at 2.38?Å resolution. Acta Crystallogr D Biol Crystallogr 67:471-9
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Sun, Ping; Tropea, Joseph E; Waugh, David S (2011) Enhancing the solubility of recombinant proteins in Escherichia coli by using hexahistidine-tagged maltose-binding protein as a fusion partner. Methods Mol Biol 705:259-74

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