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Two fast and efficient purification methods for the preparation of large amounts of proline-specific endopeptidase (PSE) [EC 3.4.21.26] from Flavobacterium meningosepticum heterologously expressed in Escherichia coli are described. Overproduction and accumulation of PSE in the periplasmic space of E. coli means that a single gel chromatography step or ion exchange chromatography step was sufficient to obtain homogenously pure PSE preparations. With these procedures, up to 490 μg of purified enzyme per g E. coli cells were obtained. The different purification methods are discussed. [ABSTRACT FROM AUTHOR]

Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.

The use of different nanoparticles (NPs) for successful encapsulation of bioactive substances is discussed. The inclusion efficiency into liposomes, acetalated dextran (Ac-Dex), and variants of poly[(lactic acid)-co-(glycolic acid)] (PLGA) NPs is analyzed after chemical degradation. Efficient inclusion of SIRT1 inhibitor Ex527 in liposomes, Ac-Dex- and PLGA-NPs is observed for all procedures used. Activity of Ex527 is demonstrated by monitoring the acetylation status of SIRT1-target p53. In contrast, small peptides are only incorporated into acid-terminated PLGA-NPs and marginally into Ac-Dex-NPs. The yield depends on peptide sequence and terminal modifications. Activity is exemplified for angiotensin II using the dynamic mass redistribution technology., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Rh2 resistance gene of barley (Hordeum vulgare) confers resistance against the scald pathogen (Rhynchosporium secalis). A high-resolution genetic map of the Rh2 region on chromosome I (7H) was established by the use of molecular markers. Tightly linked markers from this region were used to screen existing and a newly constructed yeast artificial chromosome (YAC) library of barley cv. Franka composed of 45,000 clones representing approximately two genome equivalents. Corresponding YAC clones were identified for most markers, indicating that the combined YAC library has good representation of the barley genome. The contiguous sets of YAC clones with the most tightly linked molecular markers represent entry points for map-based cloning of this resistance gene.

We describe the construction of a specific yeast artificial chromosome (YAC) library from barley (Hordeum vulgare L.) using the vector pYAC-RC. The library was generated by total digestion of high molecular weight DNA with the infrequently cutting restriction enzyme MluI. Only 10-30% of the colonies were recombinant, as visualized by red-white selection and subsequent pulsed-field gel electrophoresis analysis. About 17 000 individual recombinant YAC clones with insert sizes ranging from 50 to 700 kb, with a mean of 170 kb, were selected. No chloroplast sequences were detected and the proportion of YAC clones containing BARE-1 copia-like retroelements is about 5%. Screening of the library with a single-copy RFLP marker closely linked to the Mla locus yielded three identical clones of the same size. Insert termini of randomly chosen YAC clones were investigated with respect to their redundancy in the barley genome and compared with termini of YAC clones from an EcoRI-based YAC library, resulting in a fourfold enrichment of single-copy sequences at the MluI vector-insert junctions.

The clostripain core protein is composed of the light and heavy chain subunits linked by a nonapeptide into a single polypeptide chain [Mol. Gen. Genet. 240: 140, 1993]. Linker removal is due to autocatalytic processing yielding active heterodimeric enzyme. We have expressed mutationally altered core protein variants in the heterologous host Escherichia coli to gain further insight into the process of clostripain automaturation. In a mutationally created Cys231 --> Ser variant, heterodimer formation was largely impaired, providing molecular evidence that the capacity for automaturation is attributed to the active site cysteine, Cys231, of the native enzyme. Artificially generated deletions of the linker peptide did not prevent the formation of active enzyme. One variant gave rise to a single-chain molecule devoid of the authentic processing sites while retaining enzymatic activity. Experiments performed with linker substitution variants suggested that the efficacy of automaturation depends on a proper configuration of the linker region. According to computerized predictions, the formation of a turn-structured protein loop or hinge with hydrophilic characteristics in the linker region is probably a prerequisite for the interaction of the active site cysteine with the processing sites, Arg181 and Arg190. We propose that the clostripain linker nonapeptide serves as an important transient intramolecular inhibitor in the cellular self-defense program evolved by the natural host Clostridium histolyticum.

Clostripain-specific antibodies were used to analyse the maturation of clostripain prepro-enzyme and core protein heterologously synthesized in Escherichia coli and Bacillus subtilis. Core protein purified from E. coli cells harbouring plasmid pHM3-23 underwent calcium-dependent, self-triggered maturation. Concomitantly, the inactive form of the enzyme was converted into an active form, demonstrating the self-activation capacity of the clostripain core protein. As judged from Western blot analysis, the major portion of the protein in E. coli was degraded, presumably by the activated clostripain. The enzyme was not exported to the E. coli periplasm, either by use of the putative Clostridium histolyticum signal peptide or by use of the E. coli OmpA signal peptide. Therefore, the Gram-positive micro-organism B. subtilis was chosen as an alternative host for the expression of the prepro-enzyme and the core protein. BR 151 cells harbouring pHM7-10B secreted clostripain precursor to the growth medium and matured subsequently to the active enzyme. As only a small amount of activity was detected intracellularly, the putative C. histolyticum signal peptide was efficiently recognized by the B. subtilis secretion apparatus. Under optimized conditions, a level of 4500 U I-1 could be obtained in batch cultures.

Proline-specific endopeptidase (PSE) (EC 3.4.21.26) from Flavobacterium meningosepticum was subjected to partial amino acid sequencing. According to the peptide sequences obtained, oligonucleotides were used to amplify a PSE-specific DNA fragment of 930 bp from F. meningosepticum genomic DNA, employing the polymerase chain reaction technique. This fragment served as a molecular probe to isolate the respective gene. DNA sequencing revealed that the PSE gene consists of 2118 bp coding for a 78,634 Da protein of 705 amino acids. The coding region was cloned in different expression vectors of Escherichia coli. Transformed E. coli cells overproduce an active prolyl endopeptidase of 75,000 relative molecular mass, which is delivered to the bacterial periplasmic space. Up to 1.6 units of active prolyl endopeptidase were obtained from 1 mg E. coli cells. Furthermore, the efficient purification of active prolyl endopeptidase from the periplasm of recombinant E. coli cells is described.

Clostripain (EC 3.4.22.8) is a heterodimeric cysteine endopeptidase with strict specificity for Arg-Xaa peptidyl bonds. It is secreted by Clostridium histolyticum strains. For the first time we present evidence that both polypeptide chains of native clostripain are encoded by a single gene. DNA sequencing of two overlapping genomic DNA fragments revealed a single open reading frame (ORF) of 1581 nucleotides encoding a polypeptide of 526 amino acid residues. The ORF is preceded by canonical transcription signals and both chains of the clostripain heterodimer are completely represented by the deduced coding sequence. Most interestingly, the sequences coding for the light and the heavy chain are joined by a DNA stretch coding for a linker nonapeptide that is preceded by the C-terminal arginyl residue of the light chain and also ends with an arginyl residue. Heterologous expression of the gene in Escherichia coli yielded an enzyme capable of hydrolyzing the clostripain substrates N alpha-benzoyl-L-arginine ethyl ester (BAEE) and N-carbobenzoxy-L-arginine p-nitroanilide (Z-Arg-pNA).

Synthesis and properties of the bacterial precursor of beta-lactamase (E.C.3.5.2.6) were studied in Saccharomyces cerevisiae transformants. A protease-deficient yeast mutant was transformed with the plasmid pADH040-2 conferring high expression of the bla gene. Besides precisely processed beta-lactamase, transformed yeast cells contained mainly bla precursor up to the amount of 2% of total cellular protein. The precursor was shown to be synthesized on free polysomes in vivo but could be processed with rough microsomal membranes in a cell-free translation system. By applying an isolation procedure using high-salt conditions, the labile precursor could be separated in a native form from the mature beta-lactamase. Thereby it could be shown that the pre-beta-lactamase had virtually no enzymatic activity in contrast to the mature enzyme, which was indistinguishable from bacterial beta-lactamase. Furthermore, the precursor was highly susceptible to proteolytic degradation by trypsin under conditions which did not affect the mature enzyme. Accordingly, the protein conformation of the precursor must be substantially different from that of the authentic beta-lactamase, demonstrating that specific processing and transport of beta-lactamase is associated with directing the protein to a distinct conformation.

An abundant 0.9 kb female-specific mRNA in Schistosoma mansoni is thought to code for an egg-shell precursor protein [Bobek et al. (1986) Proc. Natl. Acad. Sci. USA 83, 5544-5548]. This gene contains two ORFs. A recombinant plasmid was constructed that expresses a fusion protein containing a glycine- and tyrosine-rich polypeptide coded for by one of these ORFs. Antisera raised against homogenates of female, but not of male, S. mansoni recognise this fusion protein, providing direct evidence that this ORF is used by S. mansoni. In comparative Western blots of S. mansoni homogenates from males and females affinity purified antibodies that react with the fusion protein react exclusively with proteins from females, recognising a 28 kDa polypeptide and a smear of immunoreactive material probably caused by oxidative crosslinking. In immunohistology, the affinity purified antibodies react with mature vitelline cells in female schistosomes. The immunoreactive material is localised in the so-called 'vitelline droplets' that are morphologically very similar to 'shell globules', known to contain egg-shell precursors, that are found in Fasciola hepatica. In situ hybridisation shows that the eggshell precursor gene is only transcribed in immature vitelline cells and has a short half-life. Taken together, these observations provide persuasive evidence that the 0.9 kb mRNA codes for an eggshell precursor.