Single chain Fab (scFab) fragment

Background  

The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display.     

Mechanistic studies of electrophilic protease inhibitors of full length hepatic C virus (HCV) NS3

The inhibition mechanism of electrophilic peptide-based protease inhibitors of full-length hepatitis C virus (HCV) NS3 has been investigated by determining the K(i)-values for a series of compounds differing in the electrophilicity and acidity of the C-terminal residue at pH-values above and below the pK(a) of the catalytic histidine (6.85) and at two different ionic strengths. Electrophilic compounds with a pentafluoroethyl ketone group showed stronger inhibition at pH 8 than pH 6, as expected for a mechanism requiring an unprotonated catalytic histidine. However, the difference was only significant at high ionic strength. In contrast, electrophilic compounds with an acidic C-terminal group or a cyclic P1 residue showed a lower inhibitory effect at pH 8 than at pH 6, inconsistent with a mechanism-based inhibition. Moreover, all electrophilic compounds had an unexpectedly strong inhibition at pH 6, when mechanism-based inhibition is unlikely. The results suggest that for some of the electrophilic compounds the reactive group may not be properly positioned in the active site and that binding of these inhibitors is a result of non-covalent interactions. The nature of these interactions is discussed.     

A new method to prevent carry-over contaminations in two-step PCR NGS library preparations

Two-step PCR procedures are an efficient and well established way to generate amplicon libraries for NGS sequencing. However, there is a high risk of cross-contamination by carry-over of amplicons from first to second amplification rounds, potentially leading to severe misinterpretation of results. Here we describe a new method able to prevent and/or to identify carry-over contaminations by introducing the K-box, a series of three synergistically acting short sequence elements. Our K-boxes are composed of (i) K1 sequences for suppression of contaminations, (ii) K2 sequences for detection of possible residual contaminations and (iii) S sequences acting as separators to avoid amplification bias. In order to demonstrate the effectiveness of our method we analyzed two-step PCR NGS libraries derived from a multiplex PCR system for detection of T-cell receptor beta gene rearrangements. We used this system since it is of high clinical relevance and may be affected by very low amounts of contaminations. Spike-in contaminations are effectively blocked by the K-box even at high rates as demonstrated by ultra-deep sequencing of the amplicons. Thus, we recommend implementation of the K-box in two-step PCR-based NGS systems for research and diagnostic applications demanding high sensitivity and accuracy.     

Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference

The discovery that the machinery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 bacterial immune system can be re-purposed to easily create deletions, insertions and replacements in the mammalian genome has revolutionized the field of genome engineering and re-invigorated the field of gene therapy. Many parallels have been drawn between the newly discovered CRISPR-Cas9 system and the RNA interference (RNAi) pathway in terms of their utility for understanding and interrogating gene function in mammalian cells. Given this similarity, the CRISPR-Cas9 field stands to benefit immensely from lessons learned during the development of RNAi technology. We examine how the history of RNAi can inform today''s challenges in CRISPR-Cas9 genome engineering such as efficiency, specificity, high-throughput screening and delivery for in vivo and therapeutic applications.     

New Mode of Energy Metabolism in the Seventh Order of Methanogens as Revealed by Comparative Genome Analysis of “Candidatus Methanoplasma termitum”

The recently discovered seventh order of methanogens, the Methanomassiliicoccales (previously referred to as “Methanoplasmatales”), so far consists exclusively of obligately hydrogen-dependent methylotrophs. We sequenced the complete genome of “Candidatus Methanoplasma termitum” from a highly enriched culture obtained from the intestinal tract of termites and compared it with the previously published genomes of three other strains from the human gut, including the first isolate of the order. Like all other strains, “Ca. Methanoplasma termitum” lacks the entire pathway for CO₂ reduction to methyl coenzyme M and produces methane by hydrogen-dependent reduction of methanol or methylamines, which is consistent with additional physiological data. However, the shared absence of cytochromes and an energy-converting hydrogenase for the reoxidation of the ferredoxin produced by the soluble heterodisulfide reductase indicates that Methanomassiliicoccales employ a new mode of energy metabolism, which differs from that proposed for the obligately methylotrophic Methanosphaera stadtmanae. Instead, all strains possess a novel complex that is related to the F₄₂₀:methanophenazine oxidoreductase (Fpo) of Methanosarcinales but lacks an F₄₂₀-oxidizing module, resembling the apparently ferredoxin-dependent Fpo-like homolog in Methanosaeta thermophila. Since all Methanomassiliicoccales also lack the subunit E of the membrane-bound heterodisulfide reductase (HdrDE), we propose that the Fpo-like complex interacts directly with subunit D, forming an energy-converting ferredoxin:heterodisulfide oxidoreductase. The dual function of heterodisulfide in Methanomassiliicoccales, which serves both in electron bifurcation and as terminal acceptor in a membrane-associated redox process, may be a unique characteristic of the novel order.