In this work we studied involvement of DNA polymerase IV (Pol IV) (encoded by the dinB gene) in stationary-phase mutagenesis in Pseudomonas putida. For this purpose we constructed a novel set of assay systems that allowed detection of different types of mutations (e.g., 1-bp deletions and different base substitutions) separately. A significant effect of Pol IV became apparent when the frequency of accumulation of 1-bp deletion mutations was compared in the P. putida wild-type strain and its Pol IV-defective dinB knockout derivative. Pol IV-dependent mutagenesis caused a remarkable increase (approximately 10-fold) in the frequency of accumulation of 1-bp deletion mutations on selective plates in wild-type P. putida populations starved for more than 1 week. No effect of Pol IV on the frequency of accumulation of base substitution mutations in starving P. putida cells was observed. The occurrence of 1-bp deletions in P. putida cells did not require a functional RecA protein. RecA independence of Pol IV-associated mutagenesis was also supported by data showing that transcription from the promoter of the P. putida dinB gene was not significantly influenced by the DNA damage-inducing agent mitomycin C. Therefore, we hypothesize that mechanisms different from the classical RecA-dependent SOS response could elevate Pol IV-dependent mutagenesis in starving P. putida cells.During the past several years our understanding of mutation mechanisms has been expanded by the discovery of a new superfamily of DNA polymerases, called the Y family (46). The Y-family polymerases have been identified in prokaryotes, archaea, and eukaryotes. Members of this superfamily are devoid of 3Ј35Ј proofreading exonuclease activity and replicate undamaged DNA with low fidelity and low processivity; many of these enzymes can bypass DNA lesions that block chain elongation by replicative DNA polymerases (21-23). According to the concept of specialized polymerases some of these polymerases are able to copy cognate lesions with high genetic fidelity (22). On the other hand, the specialized DNA polymerases are involved in mutation processes when copying noncognate DNA lesions or normal DNA.In a growth-restricting environment (e.g., during starvation), mutants arise that are able to take over bacterial populations by a process known as stationary-phase mutation (15). One widely discussed idea is that genetic adaptation of microbial populations under environmental stress might be accelerated by stress-induced activation of error-prone DNA polymerases (see, for example, references 16, 50, and 63). In Escherichia coli, two error-prone DNA polymerases, Pol V (UmuDЈC) and Pol IV (DinB), and one high-fidelity DNA polymerase, Pol II, are upregulated during the SOS response (23). SOS induction has also been shown to occur spontaneously in static bacterial populations (62). It has been recently demonstrated that errorprone DNA polymerases Pol IV and Pol V are involved in stationary-phase mutagenesis in E. coli (4,7,42). The involvement of SOS-induced polymerase...

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Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification of VLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scale able purification processes. We developed a chromatographic procedure for purification of HIV-1 gag VLPs produced in CHO cells. The clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. The majority of host cell impurities passed through the column, whereas the VLPs were eluted by a linear or step salt gradient; the major fraction of DNA was eluted prior to VLPs and particles in the range of 100-200nm in diameter could be separated into two fractions. The earlier eluted fraction was enriched with extracellular particles associated to exosomes or microvesicles, whereas the late eluting fractions contained the majority of most pure HIV-1 gag VLPs. DNA content in the exosome-containing fraction could not be reduced by Benzonase treatment which indicated that the DNA was encapsulated. Many exosome markers were identified by proteomic analysis in this fraction. We present a laboratory method that could serve as a basis for rapid downstream processing of enveloped VLPs. Up to 2000 doses, each containing 1×10(9) particles, could be processed with a 1mL monolith within 47min. The method compared to density gradient centrifugation has a 220-fold improvement in productivity.

Harnessing of a branched structure is a novel approach in the design of cell-penetrating peptides and it has provided highly efficient transfection reagents for intracellular delivery of nucleic acids. The new stearylated TP10 analogs, NickFects, condense plasmid DNA, splice correcting oligonucleotides and short interfering RNAs into stable nanoparticles with a size of 62-160nm. Such nanoparticles have a negative surface charge (-11 to -18mV) in serum containing medium and enable highly efficient gene expression, splice correction and gene silencing. One of the novel peptides, NickFect51 is capable of transfecting plasmid DNA into a large variety of cell lines, including refractory suspension and primary cells and in several cases exceeds the transfection level of commercially available reagent Lipofectamine™ 2000 without any cytotoxic side effects. Additionally we demonstrate the advantages of NickFect51 in a protein production system, QMCF technology, for expression and production of recombinant proteins in hardly transfectable suspension cells.

The rapid quantification of enveloped virus-like particles (VLPs) requires orthogonal methods to obtain reliable results. Three methods-nanoparticle tracking analysis (NTA), size-exclusion HPLC (SE-HPLC) with UV detection, and detection with multi-angle light scattering (MALS)-for quantification of enveloped VLPs have been compared, and the lower and upper limits of detection and quantification have been evaluated. NTA directly counts the enveloped VLPs, and a particle number is obtained with a lower limit of detection (LLOD) of 1.7×10part/mL and lower limit of quantification (LLOQ) of 3.4×10part/mL. SE-HPLC with UV detection was calibrated with standards characterized by NTA, and a LLOD of 6.9×10part/mL and LLOQ of 2.1×10part/mL were found. SE-HPLC with MALS does not require a pre-calibrated sample because with a spherical model based on the Rayleigh-Gans-Debye approximation, the particle concentration can be directly deduced from the scattered light. A LLOD of 4.8×10part/mL and LLOQ of 2.1×10part/mL were measured and substantially lower compared to the UV method. The absolute particle concentration measured by SE-HPLC-MALS is one order of magnitude lower compared to measurement by NTA, which is explained by the wide size distribution of an enveloped VLP suspension. The model used for evaluation of light scattering data assumes monodisperse, homogeneous, and spherical particles.