Artificial Environments for the Co-Translational Stabilization of Cell-Free Expressed Proteins

An approach for designing individual expression environments that reduce or prevent protein aggregation and precipitation is described. Inefficient folding of difficult proteins in unfavorable translation environments can cause significant losses of overexpressed proteins as precipitates or inclusion bodies. A number of chemical chaperones including alcohols, polyols, polyions or polymers are known to have positive effects on protein stability. However, conventional expression approaches can use such stabilizing agents only post-translationally during protein extraction and purification. Proteins that already precipitate inside of the producer cells cannot be addressed. The open nature of cell-free protein expression systems offers the option to include single chemicals or cocktails of stabilizing compounds already into the expression environment. We report an approach for systematic screening of stabilizers in order to improve the solubility and quality of overexpressed proteins co-translationally. A comprehensive list of representative protein stabilizers from the major groups of naturally occurring chemical chaperones has been analyzed and their concentration ranges tolerated by cell-free expression systems have been determined. As a proof of concept, we have applied the method to improve the yield of proteins showing instability and partial precipitation during cell-free synthesis. Stabilizers that co-translationally improve the solubility and functional folding of human glucosamine 6-phosphate N-acetyltransferase have been identified and cumulative effects of stabilizers have been studied.     

Transient protein expression systems in plants and their applications

The production of recombinant proteins is important in academic research to identify protein functions. Moreover, recombinant enzymes are used in the food and chemical industries, and high-quality proteins are required for diagnostic, therapeutic, and pharmaceutical applications. Though many recombinant proteins are produced by microbial or mammalian cell-based expression systems, plants have been promoted as alternative, cost-effective, scalable, safe, and sustainable expression systems. The development and improvement of transient expression systems have significantly reduced the period of protein production and increased the yield of recombinant proteins in plants. In this review, we consider the importance of plant-based expression systems for recombinant protein production and as genetic engineering tools.     

Solid-state NMR structure determination

Biological applications of solid-state NMR (SS-NMR) have been predominantly in the area of model membrane systems. Increasingly the focus has been membrane peptides and proteins. SS-NMR is able to provide information about how the peptides or proteins interact with the lipids or other peptides/proteins in the membrane, their effect on the membrane and the location of the peptides or proteins relative to the membrane surface. Recent developments in biological SS-NMR have been made possible by improvements in labelling and NMR techniques. This review discusses aligned systems and magic angle spinning techniques, bilayers and bicelles, and measurement of chemical shift anisotropy and dipolar coupling. A number of specific experiments such as cross polarization, rotational resonance, REDOR, PISEMA, MAOSS and multidimensional experiments are described. In addition to 2H, 13C and 15N, recent solid-sate 1H, 19F and 17O NMR work is discussed. Several examples of the use of SS-NMR to determine the structure of membrane peptides and proteins are given.     

Recent advances on proteins of plant terminal membranes.

Since the beginning of the 1990s, our knowledge of the protein equipment of plant membranes progresses at an accelerating pace, owing to the irruption of molecular biology tools and genetics strategies in plant biology. Map-based cloning strategies and exploration of EST databases rapidly enrich the catalog of cDNA or gene sequences expected to code for membrane proteins. The accumulation of 'putative' membrane proteins reinforces the need for structural, functional and physiological information. Indeed, ambiguities often exist concerning the association to a membrane, the membrane identity and the topology of the protein inserted in the membrane. The combination of directed mutagenesis and heterologous expression of plant genes in various systems and plant reverse genetics has opened the possibility to study molecular and physiological functions. This review will emphasize how these tools have been essential for the exciting recent discoveries on plant terminal membrane proteins. These discoveries concern a variety of transport systems for ions, organic solutes including auxin, water channels, a large collection of systems suspected to act as receptors of chemical signals, proteins thought to control vesicle trafficking and enzymatic systems.     

Sequence homology analysis of proteins by chemical cleavages: using a mono and two dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The examination of possible sequence homology in proteins using SDS-PAGE systems after chemical cleavage is described. After SDS-PAGE, the establishment of amino acid compositions, the techniques of staining gel and five different methods of chemical cleavages (cyanogen bromide, BNPS-skatole, hydroxylamine, formic acid and nitrothiocyano benzoic acid) have been used for peptide mapping studies. Potential applications of this technique are discussed from both the biochemical and immunochemical point of view.     

Protein expression profiling in chemical carcinogenesis: a proteomic-based approach

The simultaneous analysis of a wide array of proteins may provide valuable information on the activation and suppression of cellular systems at different stages of the exposure-disease continuum. In this review, results of proteomic studies in the field of toxicology are covered, focusing on the effects of chemical carcinogens. So far, alterations of highly abundant proteins have been identified which, irrespective of the wide differences in study design and technologies used, can be grossly assigned to three functional classes: proteins related to cellular stress response, inflammation, and stimulation of the immune system. It is obvious that the observed protein alterations are not causal factors in the development of chemically induced cancer but rather reflect common reactions to cellular perturbations. In order to gain deeper insights into the process of chemical carcinogenesis, the previously applied "shotgun" analyses have to be abandoned in favour of targeted proteomic approaches focusing on the accurate identification and quantification of selected proteins. Advanced analytical techniques such as selective reaction monitoring (SRM) and multiple reaction monitoring (MRM) offer this opportunity. If toxicoproteomic research moves into that direction and takes advantage of such techniques it will have the potential to contribute to the elucidation of chemical carcinogenesis.     

Chemical communication in proteobacteria: biochemical and structural studies of signal synthases and receptors required for intercellular signalling

Cell-cell communication via the production and detection of chemical signal molecules has been the focus of a great deal of research over the past decade. One class of chemical signals widely used by proteobacteria consists of N-acyl-homoserine lactones, which are synthesized by proteins related to LuxI of Vibrio fischeri and are detected by proteins related to the V. fischeri LuxR protein. A related marine bacterium, Vibrio harveyi, communicates using two chemical signals, one of which, autoinducer-2 (AI-2), is a furanone borate diester that is synthesized by the LuxS protein and detected by a periplasmic protein called LuxP. Evidence from a number of laboratories suggests that AI-2 may be used as a signal by diverse groups of bacteria, and might permit intergeneric signalling. These two families of signalling systems have been studied from the perspectives of physiology, ecology, biochemistry, and more recently, structural biology. Here, we review the biochemistry and structural biology of both acyl-homoserine-lactone-dependent and AI-2-dependent signalling systems.     

The coordination sphere of iron-sulfur clusters: lessons from site-directed mutagenesis experiments

 Cysteine is the ubiquitous ligand of iron-sulfur clusters in proteins, although chemical models have indicated that functional groups other than thiolates can coordinate iron in iron-sulfur compounds. Only a small number of naturally occurring examples of hydroxyl, histidinyl or carboxyl coordination have been clearly established but many others are suspected. Quite a few site-directed mutagenesis experiments have been aimed at replacing the cysteine ligands of iron-sulfur centers by other amino acids in various systems. The available data set shows that substituting one ligand, even by another functional residue, is very often destabilizing enough to impair cluster assembly; in some cases, the apoprotein cannot even be detected. One for one replacements have been demonstrated, but they have been so far almost exclusively confined to clusters with no more than one or two iron atoms. In contrast, changes of the cluster nuclearity or recruitment of free cysteine residues seem preferred ways for proteins containing larger clusters to cope with removal of a ligand, rather than using coordinating amino acids bearing different chemical functions. Furthermore, the possibility of replacing cysteines by other residues as ligands in iron-sulfur proteins does not uniquely depend on the ability of the cluster to accept other kinds of coordination than cysteinate; other factors such as the local flexibility of the polypeptide chain, the accessibility of the solvent and the electronic distribution on the active centers may also play a prominent role.     

A review of advanced oral drug delivery technologies facilitating the protection and absorption of protein and peptide molecules

The oral delivery of proteins and peptides is a dynamic research field despite the numerous challenges limiting their effective delivery. Successful oral delivery of proteins and peptides requires the accomplishment of three key tasks: protection of the macromolecules from degradation in the gastrointestinal tract (GIT), permeation through the intestinal barrier and absorption of molecules into the systemic circulation. Currently, no clinically useful oral formulations have been developed but several attempts have been made to overcome the challenges of low oral bioavailability resulting from poor absorption, poor permeation and enzymatic degradation of the proteins and peptides in the GIT. Present strategies attempt to provide structural protection of the proteins and peptides and improved absorption through the use of enzyme inhibitors, absorption enhancers, novel polymeric delivery systems and chemical modification. However, each of these technologies has their limitations despite showing positive results. This review attempts to discuss the physical and chemical barriers of the GIT with particular emphasis on the current approaches employed to overcome these barriers, including the evaluation of other non-parenteral routes of protein and peptide delivery. In addition, this review assimilates oral formulation strategies under development and within the clinical trial stage in relation to their benefits and drawbacks with regard to facilitating optimal protection and absorption of proteins and peptides, as well as pertinent future challenges and opportunities governing oral drug delivery.     

Chlamydomonas reinhardtii

Recombinant proteins have become more and more important for the pharmaceutical and chemical industry. Although various systems for protein expression have been developed, there is an increasing demand for inexpensive methods of large-scale production. Eukaryotic algae could serve as a novel option for the manufacturing of recombinant proteins, as they can be cultivated in a cheap and easy manner and grown to high cell densities. Being a model organism, the unicellular green alga Chlamydomonas reinhardtii has been studied intensively over the last decades and offers now a complete toolset for genetic manipulation. Recently, the successful expression of several proteins with pharmaceutical relevance has been reported from the nuclear and the chloroplastic genome of this alga, demonstrating its ability for biotechnological applications.     

The mechanism of cryoprotection of proteins by solutes

We have tested the capacity of 28 different compounds to protect lactate dehydrogenase from damage during freeze-thawing. These solutes come from very dissimilar chemical classes including sugars, polyols, amino acids, methylamines, and lyotropic salts. All the compounds tested, except NaCl, protected the enzyme, to varying degrees, from inactivation. The only characteristic that these compounds have in common, as a group, is that they have all been shown to be preferentially excluded from contact with the surface of proteins in aqueous solution. It has been demonstrated previously (via thermodynamic arguments) that this interaction of solutes with proteins leads to the stabilization of proteins in nonfrozen, aqueous systems. Conversely, those solutes, e.g., urea and guanidine HCl, that bind to proteins destabilize proteins in solution, and we have found that they also enhanced the inactivation of lactate dehydrogenase during freeze-thawing. Based on the results of our freeze-thawing experiments and a review of the theory of protein stabilization in nonfrozen, aqueous solution we propose that the cryoprotection afforded to isolated proteins by solutes can be accounted for by the fact that these solutes are preferentially excluded from contact with the protein's surface.     

Aqueous two-phase systems

Biphasic systems formed by mixing of two polymers or a polymer and a salt in water can be used for separation of cells, membranes, viruses, proteins, nucleic acids, and other biomolecules. The partitioning between the two phases is dependent on the surface properties and conformation of the materials, and also on the composition of the two-phase system. The mechanism of partitioning is, however, complex and not easily predicted. Aqueous two-phase systems (ATPS) have proven to be a useful tool for analysis of biomolecular and cellular surfaces and their interactions, fractionation of cell populations, product recovery in biotechnology, and so forth. Potential for environmental remediation has also been suggested. Because ATPS are easily scalable and are also able to hold high biomass load in comparison with other separation techniques, the application that has attracted most interest so far has been the large-scale recovery of proteins from crude feedstocks. As chemicals constitute the major cost factor for large-scale systems, use of easily recyclable phase components and the phase systems generated by a single-phase chemical in water are being studied.     

Proteinopathies--forms of neurodegenerative disorders with protein aggregation-based pathology

A number of neurodegenerative disorders have recently been coalesced into a group of proteinopathies because of the similarity of molecular mechanisms underlying their pathogenesis. A key step in the development of proteinopathies is a structural change that triggers aggregation of proteins, which are intrinsically prone to form aggregates due to their physical and chemical properties. Present review is devoted to the recent progress in the field of proteinopathies with specific focus on properties of aggregate-prone proteins, main stages of the development of molecular pathology and the role of cellular clearance systems in progression of neurodegeneration. Recent modifications in the nomenclature of neurodegenerative diseases will also be addressed.     

Microfabricated reaction and separation systems

Over the past year there have been a number of recent advances in the fields of miniaturized reaction and separation systems, including the construction of fully integrated 'lab-on-a-chip' systems. Microreactors, which initially targeted DNA-based reactions such as the polymerase chain reaction, are now used in several other chemical and biochemical assays. Miniaturized separation columns are currently employed for analyzing a wide variety of samples including DNA, RNA, proteins and cells. Although significant advances have been made at the component level, the realization of an integrated analysis system still remains at the early stages of development.     

Computational protein design

The field of computational protein design is reaching its adolescence. Protein design algorithms have been applied to design or engineer proteins that fold, fold faster, catalyze, catalyze faster, signal, and adopt preferred conformational states. Further developments of scoring functions, sampling strategies, and optimization methods will expand the range of applicability of computational protein design to larger and more varied systems, with greater incidence of success. Developments in this field are beginning to have significant impact on biotechnology and chemical biology.     

Analysis of multiple compound–protein interactions reveals novel bioactive molecules

The discovery of novel bioactive molecules advances our systems‐level understanding of biological processes and is crucial for innovation in drug development. For this purpose, the emerging field of chemical genomics is currently focused on accumulating large assay data sets describing compound–protein interactions (CPIs). Although new target proteins for known drugs have recently been identified through mining of CPI databases, using these resources to identify novel ligands remains unexplored. Herein, we demonstrate that machine learning of multiple CPIs can not only assess drug polypharmacology but can also efficiently identify novel bioactive scaffold‐hopping compounds. Through a machine‐learning technique that uses multiple CPIs, we have successfully identified novel lead compounds for two pharmaceutically important protein families, G‐protein‐coupled receptors and protein kinases. These novel compounds were not identified by existing computational ligand‐screening methods in comparative studies. The results of this study indicate that data derived from chemical genomics can be highly useful for exploring chemical space, and this systems biology perspective could accelerate drug discovery processes.     

Analysis of protein folding and function using backbone modified proteins

With the recent development of chemical and biological methods to introduce backbone modifications into the polypeptide chains of proteins, there have been a growing number of site-directed mutagenesis experiments focused on understanding the role of the polypeptide backbone in protein folding and function. The substitution of a main chain amide bond with an ester bond is now a popular mutation to investigate the role of the polypeptide backbone in ligand, binding, enzyme catalysis, and protein folding. Here we review the results of studies on some 25 ester-bond containing analogues from nine different protein systems. The structural, thermodynamic, and functional consequences of introducing backbone amide- to ester-bond mutations into these protein systems are discussed.     

Amyloids and prions in plants: Facts and perspectives

Amyloids represent protein fibrils that have highly ordered structure with unique physical and chemical properties. Amyloids have long been considered lethal pathogens that cause dozens of incurable diseases in humans and animals. Recent data show that amyloids may not only possess pathogenic properties but are also implicated in the essential biological processes in a variety of prokaryotes and eukaryotes. Functional amyloids have been identified in archaea, bacteria, fungi, and animals, including humans. Plants are one of the most poorly studied groups of organisms in the field of amyloid biology. Although amyloid properties have not been shown under native conditions for any plant protein, studies demonstrating amyloid properties for a set of plant proteins in vitro or in heterologous systems in vivo have been published in recent years. In this review, we systematize the data on the amyloidogenic proteins of plants and their functions and discuss the perspectives of identifying novel amyloids using bioinformatic and proteomic approaches.     

CAMRA: Chemical shift based computer aided protein NMR assignments

A suite of programs called CAMRA (Computer Aided Magnetic Resonance Assignment) has been developed for computer assisted residue-specific assignments of proteins. CAMRA consists of three units: ORB, CAPTURE and PROCESS. ORB predicts NMR chemical shifts for unassigned proteins using a chemical shift database of previously assigned homologous proteins supplemented by a statistically derived chemical shift database in which the shifts are categorized according to their residue, atom and secondary structure type. CAPTURE generates a list of valid peaks from NMR spectra by filtering out noise peaks and other artifacts and then separating the derived peak list into distinct spin systems. PROCESS combines the chemical shift predictions from ORB with the spin systems identified by CAPTURE to obtain residue specific assignments. PROCESS ranks the top choices for an assignment along with scores and confidence values. In contrast to other auto-assignment programs, CAMRA does not use any connectivity information but instead is based solely on matching predicted shifts with observed spin systems. As such, CAMRA represents a new and unique approach for the assignment of protein NMR spectra. CAMRA will be particularly useful in conjunction with other assignment methods and under special circumstances, such as the assignment of flexible regions in proteins where sufficient NOE information is generally not available. CAMRA was tested on two medium-sized proteins belonging to the chemokine family. It was found to be effective in predicting the assignment providing a database of previously assigned proteins with at least 30% sequence identity is available. CAMRA is versatile and can be used to include and evaluate heteronuclear and three-dimensional experiments.