Catalysts Confined in Programmed Framework Pores Enable New Transformations and Tune Reaction Efficiency and Selectivity.

Controlling chemical reactions in porous heterogeneous catalysts is a tremendous challenge because of the difficulty in producing uniform active sites that can be tuned with precision. However, analogous to enzymes, when a catalytic pocket provides complementary close contacts and favorable intermolecular interactions with the reaction participants, the reaction efficiency and selectivity may be tuned. Here, we report an isoreticular family of catalysts based on the multicomponent metal-organic framework MUF-77.

Crystal structures of the viral protease Npro imply distinct roles for the catalytic water in catalysis.

Npro is a key effector protein of pestiviruses such as bovine viral diarrhea virus and abolishes host cell antiviral defense mechanisms. Synthesized as the N-terminal part of the viral polyprotein, Npro releases itself via an autoproteolytic cleavage, triggering its immunological functions. However, the mechanisms of its proteolytic action and its immune escape were unclear.

FlgM as a secretion moiety for the development of an inducible type III secretion system.

Regulation and assembly of the flagellar type III secretion system is one of the most investigated and best understood regulational cascades in molecular biology. Depending on the host organism, flagellar morphogenesis requires the interplay of more than 50 genes. Direct secretion of heterologous proteins to the supernatant is appealing due to protection against cellular proteases and simplified downstream processing.

Dissection of an old protein reveals a novel application: domain D of Staphylococcus aureus Protein A (sSpAD) as a secretion--tag.

Background: Escherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous.

Refolding of Npro fusion proteins.

The autoprotease Npro significantly enhances expression of fused peptides and proteins and drives the formation of inclusion bodies during protein expression. Upon refolding, the autoprotease becomes active and cleaves itself specifically at its own C-terminus releasing the target protein with its authentic N-terminus. Npro wild-type and its mutant EDDIE, respectively, were fused N-terminally to the model proteins green fluorescent protein, staphylococcus Protein A domain D, inhibitory peptide of senescence-evasion-factor, and the short 16 amino acid peptide pep6His.

A novel Ecotin-Ubiquitin-Tag (ECUT) for efficient, soluble peptide production in the periplasm of Escherichia coli.

Background: Many protocols for recombinant production of peptides and proteins include secretion into the periplasmic space of Escherichia coli, as they may not properly fold in the cytoplasm. If a signal peptide is not sufficient for translocation, a larger secretion moiety can instead be fused to the gene of interest. However, due to the covalent linkage of the proteins, a protease recognition site needs to be introduced in between, altering the N-terminus of the product.

N(pro) fusion technology to produce proteins with authentic N termini in E. coli.

We describe a prokaryotic expression system using the autoproteolytic function of N(pro) from classical swine fever virus. Proteins or peptides expressed as N(pro) fusions are deposited as inclusion bodies. On in vitro refolding by switching from chaotropic to kosmotropic conditions, the fusion partner is released from the C-terminal end of the autoprotease by self-cleavage, leaving the target protein with an authentic N terminus.

HB tag modules for PCR-based gene tagging and tandem affinity purification in Saccharomyces cerevisiae.

We have recently developed the HB tag as a useful tool for tandem-affinity purification under native as well as fully denaturing conditions. The HB tag and its derivatives consist of a hexahistidine tag and a bacterially-derived in vivo biotinylation signal peptide, which support sequential purification by Ni2+ -chelate chromatography and binding to immobilized streptavidin. To facilitate tagging of budding yeast proteins with HB tags, we have created a series of plasmids with various selectable markers.

A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivocross-linking.

Tandem affinity strategies reach exceptional protein purification grades and have considerably improved the outcome of mass spectrometry-based proteomic experiments. However, current tandem affinity tags are incompatible with two-step purification under fully denaturing conditions. Such stringent purification conditions are desirable for mass spectrometric analyses of protein modifications as they result in maximal preservation of posttranslational modifications.