Publications by authors named "Schumann W"

Guanylate-binding proteins (GBPs) are essential interferon-γ-activated large GTPases that play a crucial role in host defense against intracellular bacteria and parasites. While their protective functions rely on protein polymerization, our understanding of the structural intricacies of these multimerized states remains limited. To bridge this knowledge gap, we present dimer models for human GBP1 (hGBP1) and murine GBP2 and 7 (mGBP2 and mGBP7) using an integrative approach, incorporating the crystal structure of hGBP1's GTPase domain dimer, crosslinking mass spectrometry, small-angle X-ray scattering, protein-protein docking, and molecular dynamics simulations.

View Article and Find Full Text PDF

Choosing fusion tags to enhance the recombinant protein levels in the cytoplasm of Bacillus subtilis has been limited. Our previous study demonstrated that His-tag at the N-terminus could increase the expression levels of the low-expression gene egfp, while significantly reducing the high-expression genes gfp+ and bgaB in the cytoplasm of B. subtilis.

View Article and Find Full Text PDF

The IPTG-inducible promoter family, Pgrac, allows high protein expression levels in an inducible manner. In this study, we constructed IPTG-inducible expression vectors containing strong Pgrac promoters that allow integration of the transgene at either the amyE or lacA locus or both loci in Bacillus subtilis. Our novel integrative expression vectors based on Pgrac promoters could control the repression of protein production in the absence and the induction in the presence of an inducer, IPTG.

View Article and Find Full Text PDF

The influence of fusion tags to produce recombinant proteins in the cytoplasm of is not well-studied as in . This study aimed to investigate the influence of His-tags with different codons on the protein production levels of the high expression gene () and low expression gene () in the cytoplasm of cells. We used three different N-terminal His-tags, M-6xHis, MRGS-8xHis and MEA-8xHis, to investigate their effects on the production levels of GFP variants under the control of the P212 in .

View Article and Find Full Text PDF

Protein disorder and aggregation play significant roles in the pathogenesis of numerous neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. The end products of the aggregation process in these diseases are highly structured amyloid fibrils. Though in most cases, small, soluble oligomers formed during amyloid aggregation are the toxic species.

View Article and Find Full Text PDF
Article Synopsis
  • Inducer-free integrative vectors are utilized for creating industrial strains but face issues with leaky expression when using strong promoters for recombinant protein production.
  • By developing strong IPTG-inducible promoters with operators, researchers created vectors that integrate into specific genome loci, successfully reducing unwanted gene expression and allowing for better control of β-galactosidase gene repression.
  • The study found that these new vectors could achieve significant levels of protein expression without inducers, demonstrating the effectiveness of the P promoter family for producing recombinant proteins in industrial applications.
View Article and Find Full Text PDF

Expression and secretion of recombinant proteins in the endotoxin-free bacterium, Bacillus subtilis, has been thoroughly studied, but overexpression in the cytoplasm has been limited to only a few proteins. Here, we used the robust IPTG-inducible promoter, Pgrac212, to overexpress human rhinovirus 3C protease (HRV3C) in the cytoplasm of B. subtilis cells.

View Article and Find Full Text PDF

Background: Besides Escherichia coli, Bacillus subtilis is an important bacterial species for the production of recombinant proteins. Recombinant genes are inserted into shuttle expression vectors which replicate in both E. coli and in B.

View Article and Find Full Text PDF

All organisms developed genetic programs to allow their survival under stressful conditions. In most cases, they increase the amount of a specific class of proteins which deal with the stress factor and allow cells to adapt to life-threatening conditions. One class of stress proteins are the heat shock proteins (HSPs) the amount of which is significantly increased after a sudden temperature rise.

View Article and Find Full Text PDF

Surface display using spores of Bacillus subtilis is widely used to anchor antigens and enzymes of different sources. One open question is whether anchored proteins are able to form disulfide bonds. To answer this important question, we anchored the Escherichia coli alkaline phosphatase PhoA on the spore surface using two different surface proteins, CotB and CotZ.

View Article and Find Full Text PDF

Background: In general, fusion of recombinant genes to strong inducible promoters allowing intracellular expression in Bacillus subtilis is a two-step process. The ligation products are transformed into Escherichia coli, followed by identification of the correct plasmid, and this plasmid is subsequently transformed into B. subtilis.

View Article and Find Full Text PDF

Transposons are important tools to inactivate chromosomal genes followed by a correlation with a particular phenotype or genotype. Here we demonstrated the development of a special type of genetically engineered transposon carrying an outward-directed inducible promoter in order to allow transcription of nearby genes. We have modified the mariner transposon TnYLB able to transpose in B.

View Article and Find Full Text PDF

The method of surface display allows the fusion of passenger proteins to a carrier protein displayed on the outside of bioparticles such as spores. Here, we used spores of Bacillus subtilis, the outer surface proteins CotB, CotC, and CotG as carrier and the amyQ-encoded α-amylase and GFPuv as passenger proteins. The different translational fusions were fused to two different IPTG-inducible promoters, and the regulated expression level of both passenger proteins were measured in relation to the inducer concentration added to sporulating cells.

View Article and Find Full Text PDF

Different mRNA stabilizing elements including the 3'-stem-loop, the ribosome binding sites (RBS), the 5'-stem-loop and the spacer region between the RBS and the 5'-stem-loop were analysed in detail to increase mRNA stability resulting in enhanced expression of heterologous proteins. In addition, in combination with mRNA stabilizing elements, we propose a new class of 5'-mRNA controllable stabilizing element (CoSE) which is composed of a transcriptional operator such as lacO of the Escherichia coli lac operon and a suitable RBS followed by an optimal spacer length. Such a CoSE allowed Bacillus subtilis cells to synthesize extraordinary stable transcripts with a half-life of the model bgaB reporter transcript (codes for an β-galactosidase gene derived from Bacillus stearothermophilus) of more than 60 min.

View Article and Find Full Text PDF

The key components of the major secretion pathway in bacteria, the Sec pathway, are the proteins SecA, an ATPase that generates the energy required for protein translocation, and the heterotrimeric protein complex SecYEG, which functions as the preprotein-conducting channel through the cytoplasmic membrane, named translocon. Overexpression of exoproteins can cause jamming of the membrane, e.g.

View Article and Find Full Text PDF

BACKGROUND: For increasing allograft tendon safety in reconstructive surgery, an effective sterilization method achieving sterility assurance including viruses without impairing the grafts properties is needed. Fractionated Electron Beam (Ebeam) has shown promising in vitro results. The proof of sufficient virus inactivation is a central part of the process validation.

View Article and Find Full Text PDF

Four different mechanisms have evolved in eubacteria to comply with changes in the environmental temperature. The underlying genetic mechanisms regulate gene expression at transcriptional, translational and posttranslational level. The high temperature response (HTR) is a reaction on increases in temperature and is mainly used by pathogenic bacteria when they enter their mammalian host.

View Article and Find Full Text PDF

In an attempt to identify substrate proteins of the FtsH metalloprotease involved in stage 0 of sporulation in Bacillus subtilis, the proteome of an ftsH wild-type strain was compared to that of an ftsH null mutant by the 2D gel technique. One of the most abundant proteins identified in the absence of ftsH turned out to be Spo0M, a sporulation control protein of stage 0. Using transcriptional fusion between the spo0M promoter and the bgaB reporter gene, expression analysis did not reveal any influence of FtsH on spo0M transcription, suggesting that FtsH might have a negative effect on the stability of Spo0M due to its proteolytic activity on Spo0M.

View Article and Find Full Text PDF

Transcription efficiency of inducible promoters remains a bottleneck in recombinant protein production in Bacillus subtilis cells. Here, we present experimental data how to generate strong IPTG-inducible promoters by optimization of nucleotides at the conserved regions of the groESL promoter including the UP element, the -35, -15, -10 and the +1 region. Combination of these changes into one promoter enhanced the amount of recombinant proteins accumulating intracellularly up to about 30% of the total cellular protein.

View Article and Find Full Text PDF

Bacillus subtilis codes for two putative sortases, YhcS and YwpE, and two surface proteins, YhcR and YfkN, harboring sorting motifs supposed to be recognized by the putative sortase(s). However, there is no experimental evidence to show a direct link between these sortases and sorting sequences. To study the role of these two putative sortases on displaying YhcR and YfkN on the cell wall, expression of yhcS and ywpE was analyzed by transcriptional fusions and by Northern blot.

View Article and Find Full Text PDF

Using published plasmid vectors containing the bgaB gene encoding a heat-stable beta-galactosidase, we have been unable to fuse strong promoters to this reporter gene. In addition, we could not analyze the promoter strength by a plate assay. Therefore, we constructed an Escherichia coli-Bacillus subtilis shuttle vector to allow the cloning of strong promoters and their rapid analysis in B.

View Article and Find Full Text PDF

In this paper, we present a detailed investigation into the suitability of atomic force microscopy (AFM) cantilevers with integrated deflection sensor and micro-actuator for imaging of soft biological samples in fluid. The Si cantilevers are actuated using a micro-heater at the bottom end of the cantilever. Sensing is achieved through p-doped resistors connected in a Wheatstone bridge.

View Article and Find Full Text PDF

About 2% of the Bacillus subtilis genes are subject to regulation by riboswitch-controlled mechanisms. One of them is the L-lysine-dependent lysC gene which is turned on when the L-lysine concentration within the cytoplasm is low. In the presence of a high L-lysine concentration, only a 0.

View Article and Find Full Text PDF

The targeting and anchoring of heterologous proteins and peptides to the outer surface of bacteriophages and cells is becoming increasingly important, and has been employed as a tool for fundamental and applied research in microbiology, molecular biology, vaccinology, and biotechnology. Less known are endospores or spores produced by some Gram-positive species. Spores of Bacillus subtilis are surrounded by a spore coat on their outside, and a few proteins have been identified being located on the outside layer and have been successfully used to immobilize antigens and some other proteins and enzymes.

View Article and Find Full Text PDF

Unlabelled: We are developing a methodology for the noninvasive imaging of glucose transport in vivo with PET and (18)F-labeled 6-fluoro-6-deoxy-d-glucose ((18)F-6FDG), a tracer that is transported but not phosphorylated. To validate the method, we evaluated the biodistribution of (18)F-6FDG to test whether it is consistent with the known properties of glucose transport, particularly with regard to insulin stimulation of glucose transport.

Methods: Under glucose clamp conditions, rats were imaged at the baseline and under conditions of hyperinsulinemia.

View Article and Find Full Text PDF