Affinity cross-flow filtration: purification of IgG with a novel protein a affinity matrix prepared from two-dimensional protein crystals

In this article, we describe the use of 1- to 2-mum sized affinity microparticles for the isolation and purification of IgG from artificial IgG-human serum albumin mixtures and clarified hybridoma cell culture supernatants by affinity cross-flow filtration. Affinity microparticles were prepared from cell wall fragments of Clostridium thermohydrosulfuricum L111-69, in which the peptidoglycan-containing layer was completely covered with a hexagonally ordered S-layer lattice. After crosslinking the S-layer protein with glutaraldehyde, carboxyl groups from acidic amino acids were activated with carbodiimide and used for immobilization of Protein. A. Quantitative determination confirmed that Protein A molecules formed a monomolecular layer on the outermost surface of the S-layer lattice. Affinity microparticles were found to withstand high centrifugal and shear forces and revealed no Protein A leakage or S-layer protein release under cross-flow conditions between pH 2 to 12. The IgG-binding capacity of affinity microparticles was investigated under crossflow conditions and compared with that obtained in batch adsorption processes. (c) 1994 John Wiley & Sons, Inc.     

A synthetic medium for continuous culture of the S-layer carrying Bacillus stearothermophilus PV 72 and studies on the influence of growth conditions on cell wall properties

Bacterial cell surface layers (S-layers) which show a crystalline structure, defined pores, and a regular arrangement of functioal groups can be used for production of isoporous ultrafiltration membranes and as a matrix for immobilization of macromolecules. S-layer-carrying cell wall fragments from thermophilic Bacillaceae possess an extremely thin peptidoglycan-containing layer with pores larger than those in the S-layer lattice. Thus, they can directly be used for biotechnological applications, when an S-layer protein pool is stored in the rigid cell wall layer which is released during cell wall preparation, forming an inner S-layer. In the present study, a synthetic medium for Bacillus stearothermophilus PV 72 was developed by applying the pulse and shift technique with the aim to produce cell wall fragments with before-mentioned properties by varying the growth conditions in condtinuous culture. The organism was grown at 57 degrees C in a bioreactor with 1 L working volume equipped with exhaust gas analysis and connected to a PC-based process control system. Biomass concentration was 2.2 g/L out of 8 g/L glucose at a dilution rate of 0.3 h(-1), giving a biomass productivity of 0.66 g/L h. Although the organism was grown under different conditions, no change in peptidoglycan composition, extent of peptidoglycan crosslinking, and content of secondary cell wall polymers was observed. The amount of S-layer protein pool stored in the rigid cell wall layer and the autolytic activity depended mainly on the specific growth rate. Cell wall fragments with properties required for ultrafiltration membrane production could be produced by parameter settings in continuous culture. (c) 1995 John Wiley & Sons, Inc.     

Nearest-neighbor classifier as a tool for classification of protein families

Knowledge about protein function is essential in understanding the biological processes. A specific class or family of protein shares common structural and chemical properties amongst its member sequences. The set of properties that display its unique characteristics for clearly classifying a protein sequence into its corresponding protein family needs to be studied. Our study of these important properties conducted on four major classes of proteins namely Globins, Homeoboxes, Heat Shock proteins (HSP) and Kinase have shown that frequency of twenty naturally occurring amino acids, hydrophobic content of protein, molecular weight of protein, isoelectric point of protein, secondary structure composition of amino acid residues as helices, coils and sheets and the composition of helices, coils and sheets in the secondary structure topology plays a significant role in correctly classifying the protein into its corresponding class or family as indicated by the overall efficiency of Nearest Neighbor Classifier as 84.92%.     

Optical trap as a tool for studying motor proteins

The review briefs the optical trap method, a modern experimental tool based on the recently discovered ability of light to trap and hold micron and submicron particles in a focused beam. The physical principle underlying the optical trap and the opportunities that it provides for studying the molecular nature of biological motility are considered. Several studies into the physical characteristics and functions of single motor protein molecules performed using the optical trap and recording nanometer displacements and piconewton forces are analyzed.     

Rapid screening for putative exported proteins from staphylococcus aureus using alkaline phosphatase as a reporter molecule

Staphylococcus aureus causes a wide range of infections in humans, ranging from superficial skin infections to the more serious toxin-mediated diseases such as toxic shock syndrome. Owing to the increasing resistance of this bacterium to a wide range of antibiotics, the need to determine the virulence factors involved in infection is becoming more important as these molecules are potential therapeutic targets. In this study, we have screened for putative exported proteins from S. aureus on the basis that these proteins are likely to be the first point of contact between the bacterium and host during infection. We have constructed gene fusions between S. aureus DNA and a truncated version of the Escherichia coli phoA gene, and we report on the characterization of the recombinants exhibiting alkaline phosphatase activity. As well as known S. aureus proteins, we have identified a number of putative open reading frames that encode proteins similar to those from nonstaphylococcal species and also unique proteins that do not have any homologues on the current databases.     

Establishing the yeast Saccharomyces cerevisiae as a system for expression of human proteins on a proteome-scale

Structural genomics requires the application of a standardised process for overexpression of soluble proteins that allows high-throughput purification and analysis of protein products. We have developed a highly parallel approach to protein expression, including the simultaneous expression screening of a large number of cDNA clones in an appropriate vector system and the use of a protease-deficient host strain. A set of 221 human genes coding for proteins of various sizes with unknown structures was selected to evaluate the system. We transferred the cDNAs from an E. coli vector to the yeast expression vector by recombinational cloning, avoiding time-consuming recloning steps and the use of restriction enzymes in the cloning process. The subcloning yield was 95%, provided that a PCR fragment of the correct size could be obtained. Sixty percent of these proteins were expressed as soluble products at detectable levels and 48% were successfully purified under native conditions using the His₆ tag fusion.The advantages of the developed yeast-based expression system are the ease of manipulation and cultivation of S. cerevisiae in the same way as with prokaryotic hosts and the ability to introduce post-translational modifications of proteins if required, thus being an attractive system for heterologous expression of mammalian proteins. The expression clones selected in this screening process are passed on to the fermentation process in order to provide milligram amounts of proteins for structure analysis within the Berlin Protein Structure Factory. All data generated is stored in a relational database and is available on our website(http://www.proteinstrukturfabrik.de).     

Immortalized human hepatocytes as a tool for the study of hepatocytic (de-)differentiation

Primary human hepatocytes were immortalized by stable transfection with a recombinant plasmid containing the early region of simian virus (SV) 40. The cells were cultured in serum-free, hormonally defined medium during the immortalization procedure. Foci of dividing cells were seen after 3 months. Albumin- and fibrinogen-secreting cells were selected and cloned by limiting dilution to obtain homologous cell populations. The established IHH (immortalized human hepatocyte) cell lines were evaluated for their usefulness in studying the regulation of cell growth and of certain differentiated hepatocyte functions.IHH cells retain several differentiated features of normal hepatocytes. They display albumin secretion at a level comparable to cultured primary human hepatocytes (30 µg albumin/ml per day). A portion of the IHH cells are polarized, forming bile canaliculi-like vacuoles where exogeneous organic anions accumulate. The multidrug resistance (MDR) P-glycoprotein, known to be localized at the canalicular membrane, is also present in these vacuoles. The polarized features allowed the use of IHH cells for the study of localization of the newly characterized multidrug resistance protein MRP1. The homologues of MRP were found in hepatocytes, MRP1 and MRP2 (cMOAT), both functioning in ATP-dependent excretion of anionic conjugates. In differentiated hepatocytes, MRP1 expression is extremely low. In contrast, MRP1 is highly expressed in proliferating IHH cells, where it is localized in lateral membranes. A highly differentiated feature of short-term cultured primary hepatocytes which is not detectable in IHH cells is active uptake of the bile salt taurocholate. Furthermore, IHH cells secrete triglyceride (TG)-rich lipoproteins, apolipoprotein B (0.6 µg/ml per day), and apolipoprotein A-I (1 µg/ml per day). However, they secrete apoB-containing TG-rich lipoproteins mainly in the LDL density range, while short-term cultured primary hepatocytes mainly secrete TG-rich lipoproteins in the VLDL density range.In conclusion, functions that are rapidly lost in short-term hepatocyte cultures are, in general, not displayed by IHH cells. Immortalized human hepatocytes provide a valuable tool for studying the regulation of hepatocyte proliferation-related phenomena.     

Evidence for the glycoprotein nature of the crystalline cell wall surface layer of Bacillus stearothermophilus strain NRS2004/3a

The surface layer of Bacillus stearothermophilus strain NRS2004/3a was isolated and chemically characterized. The results of these initial studies lead to the conclusion that the cell surface protein is glycosylated.     

Mass spectrometry-based immunoassays for the next phase of clinical applications

Recent applications of affinity mass spectrometry into clinical laboratories brought a renewed interest in immunoaffinity mass spectrometry as a more specific affinity method capable of selectively targeting and studying protein biomarkers. In mass spectrometry-based immunoassays, proteins are affinity retrieved from biological samples via surface-immobilized antibodies, and are then detected via mass spectrometric analysis. The assays benefit from dual specificity, which is brought about by the affinity of the antibody and the protein mass readout. The mass spectrometry aspect of the assays enables single-step detection of protein isoforms and their individual quantification. This review offers a comprehensive review of mass spectrometry-based immunoassays, from historical perspectives in the development of the immunoaffinity mass spectrometry, to current applications of the assays in clinical and population proteomic endeavors. Described in more detail are two types of mass spectrometry-based immunoassays, one of which incorporates surface plasmon resonance detection for protein quantification. All mass spectrometry-based immunoassays offer high-throughput targeted protein investigation, with clear implications in clinical research, encompassing biomarker discovery and validation, and in diagnostic settings as the next-generation immunoassays.     

LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan

The extracellular matrix glycosaminoglycan hyaluronan (HA) is an abundant component of skin and mesenchymal tissues where it facilitates cell migration during wound healing, inflammation, and embryonic morphogenesis. Both during normal tissue homeostasis and particularly after tissue injury, HA is mobilized from these sites through lymphatic vessels to the lymph nodes where it is degraded before entering the circulation for rapid uptake by the liver. Currently, however, the identities of HA binding molecules which control this pathway are unknown. Here we describe the first such molecule, LYVE-1, which we have identified as a major receptor for HA on the lymph vessel wall. The deduced amino acid sequence of LYVE-1 predicts a 322-residue type I integral membrane polypeptide 41% similar to the CD44 HA receptor with a 212-residue extracellular domain containing a single Link module the prototypic HA binding domain of the Link protein superfamily. Like CD44, the LYVE-1 molecule binds both soluble and immobilized HA. However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels. Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels themselves.     

MALDI-TOF mass spectrometry as a tool for differentiation of invasive and noninvasive Streptococcus pyogenes isolates

A novel mass spectral fingerprinting and proteomics approach using MALDI-TOF MS was applied to detect and identify protein biomarkers of group A Streptococcus (GAS) strains. Streptococcus pyogenes ATCC 700294 genome strain was compared with eight GAS clinical isolates to explore the ability of MALDI-TOF MS to differentiate isolates. Reference strains of other bacterial species were also analyzed and compared with the GAS isolates. MALDI preparations were optimized by varying solvents, matrices, plating techniques, and mass ranges for S. pyogenes ATCC 700294. Spectral variability was tested. A subset of common, characteristic, and reproducible biomarkers in the range of 2000-14 000 Da were detected, and they appeared to be independent of the culture media. Statistical analysis confirmed method reproducibility. Random Forest analysis of all selected GAS isolates revealed differences among most of them, and summed spectra were used for hierarchical cluster analysis. Specific biomarkers were found for each strain, and invasive GAS isolates could be differentiated. GAS isolates from cases of necrotizing fasciitis were clustered together and were distinct from isolates associated with noninvasive infections, despite their sharing the same emm type. Almost 30% of the biomarkers detected were tentatively identified as ribosomal proteins.     

GFP‐SCFV: Expression and possible applications as a tool for experimental investigations of atherosclerosis

Experimental studies on atherosclerosis are crucial for investigating its pathophysiology, defining new therapeutic targets, and developing new drugs and diagnostic tools. Thus, many imaging markers have been developed and introduced in experimental studies. The main advantage of these new tools is that they allow the noninvasive diagnosis of atherosclerotic vascular disease. Here, we describe the cloning, expression, purification, and stabilization of a chimeric protein specifically designed to probe cells and tissues for the presence of LDL(?), a relevant marker of atherosclerosis. The DNA sequence that encodes the anti‐LDL(?) scFv, previously obtained from a hybridoma secreting an anti‐LDL(?) monoclonal antibody, was inserted into the bacterial vector pET‐28a(+) in tandem with a DNA sequence encoding GFP. The recombinant protein was expressed in high yields in E. coli as inclusion bodies. The applicability of GFP‐scFv was assessed by ELISA, which determined its affinity for LDL(?) and confocal microscopy, that showed macrophage uptake of the protein along with LDL(?). In conclusion, our data suggest that the anti‐LDL(?) GFP‐scFv chimeric protein could be useful in studies on atherogenesis as well as for developing diagnostic tools for atherosclerosis. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1206–1213, 2014     

Continuous metadynamics in essential coordinates as a tool for free energy modelling of conformational changes

Modelling of conformational changes in biopolymers is one of the greatest challenges of molecular biophysics. Metadynamics is a recently introduced free energy modelling technique that enhances sampling of configurational (e.g. conformational) space within a molecular dynamics simulation. This enhancement is achieved by the addition of a history-dependent bias potential, which drives the system from previously visited regions. Discontinuous metadynamics in the space of essential dynamics eigenvectors (collective motions) has been proposed and tested in conformational change modelling. Here, we present an implementation of two continuous formulations of metadynamics in the essential subspace. The method was performed in a modified version of the molecular dynamics package GROMACS. These implementations were tested on conformational changes in cyclohexane, alanine dipeptide (terminally blocked alanine, Ace-Ala-Nme) and SH3 domain. The results illustrate that metadynamics in the space of essential coordinates can accurately model free energy surfaces associated with conformational changes. Figure The conformational free energy surface of cyclohexane in the space of the two most intensive collective motions.

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Yeast as a tool for characterizing mono-ADP-ribosyltransferase toxins

The emergence of bacterial antibiotic resistance poses a significant challenge in the pursuit of novel therapeutics, making new strategies for drug discovery imperative. We have developed a yeast growth-defect phenotypic screen to help solve this current dilemma. This approach facilitates the identification and characterization of a new diphtheria toxin (DT) group, ADP-ribosyltransferase toxins from pathogenic bacteria. In addition, this assay utilizes Saccharomyces cerevisiae , a reliable model for bacterial toxin expression, to streamline the identification and characterization of new inhibitors against this group of bacterial toxins that may be useful for antimicrobial therapies. We show that a mutant of the elongation factor 2 target protein in yeast, G701R, confers resistance to all DT group toxins and recovers the growth-defect phenotype in yeast. We also demonstrate the ability of a potent small-molecule toxin inhibitor, 1,8-naphthalimide (NAP), to alleviate the growth defect caused by toxin expression in yeast. Moreover, we determined the crystal structure of the NAP inhibitor–toxin complex at near-atomic resolution to provide insight into the inhibitory mechanism. Finally, the NAP inhibitor shows therapeutic protective effects against toxin invasion of mammalian cells, including human lung cells.     

Loofa (Luffa cylindrica) sponge: Review of development of the biomatrix as a tool for biotechnological applications

The review discusses the development of loofa sponge (Luffa cylindrica) as a biotechnological tool and the diversity of applications in which it has been successfully used since it was first reported as a matrix for the immobilization of microbiological cells in 1993. The fibro‐vascular reticulated structure, made up of an open network of random lattices of small cross‐sections coupled with very high porosity (79–93%), having very low density (0.02–0.04 g/cm³), and high specific pore volume (21–29 cm³/g), has the characteristics of a carrier/scaffold well‐suited for cell immobilization. This has been confirmed through the immobilization of cells of diverse types, including filamentous and microalgae, fungi, bacteria, yeasts, higher plants, and human and rat hepatocytes. The cells immobilized in loofa sponge have performed well and better than free suspended cells and those immobilized in conventionally used natural and synthetic polymeric materials for the production of ethanol, organic acids, enzymes, and secondary metabolites. The loofa‐immobilized cell systems have been efficiently used for the treatment of wastewaters containing toxic metals, dyes, and chlorinated compounds, and the technology has been used to develop biofilms for the remediation of domestic and industrial wastewaters rich in inorganic and organic matter. In addition, three‐dimensional loofa sponge scaffolds for hepatocyte culture have been suggested to have the potential for development into a bioartificial liver device. Loofa sponge is a cost‐effective, eco‐friendly, and easy to handle matrix that has been used successfully as a biotechnological tool in a variety of systems, purposes, and applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:573–600, 2013     

Combined detection of Chlamydia trachomatis-specific antibodies against the 10 and 60-kDa heat shock proteins as a diagnostic tool for tubal factor infertility: Results from a case-control study in Cameroon

A case-control study was conducted to determine the diagnostic value of Chlamydia trachomatis-associated anti-Chsp10 and/or anti-Chsp60 antibodies in the detection of secondary infertility. There were significant associations between C. trachomatis infection and infertility (p<0.01), and between C. trachomatis-specific anti-Chsp10 or anti-Chsp60 antibodies and secondary infertility (p<0.001). A significant correlation was found between anti-Chsp10 and anti-Chsp60 titers (p<0.01). The detection of either C. trachomatis-associated anti-Chsp10 or anti-Chsp60 antibodies cumulatively allowed specific diagnosis of secondary infertility (57.4% sensitivity, 75.5% specificity).     

DNA barcoding as a tool for coral reef conservation

DNA Barcoding (DBC) is a method for taxonomic identification of animals that is based entirely on the 5′ portion of the mitochondrial gene, cytochrome oxidase subunit I (COI-5). It can be especially useful for identification of larval forms or incomplete specimens lacking diagnostic morphological characters. DBC can also facilitate the discovery of species and in defining “molecular taxonomic units” in problematic groups. However, DBC is not a panacea for coral reef taxonomy. In two of the most ecologically important groups on coral reefs, the Anthozoa and Porifera, COI-5 sequences have diverged too little to be diagnostic for all species. Other problems for DBC include paraphyly in mitochondrial gene trees and lack of differentiation between hybrids and their maternal ancestors. DBC also depends on the availability of databases of COI-5 sequences, which are still in early stages of development. A global effort to barcode all fish species has demonstrated the importance of large-scale coordination and is yielding promising results. Whether or not COI-5 by itself is sufficient for species assignments has become a contentious question; it is generally advantageous to use sequences from multiple loci.     

CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification

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