Explore the vast range of titles available.

The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity and structural conformity, in order to validate the recombinant production and purification process. Therefore, this review aims at providing succinct information on the rational purification design of recombinant proteins produced in Escherichia coli, specifically the tagging purification, as well as on accessible tools for evaluating and optimizing protein quality. The classical techniques for structural protein characterization—denaturing protein gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), dynamic light scattering (DLS) and circular dichroism (CD)—are revisited with focus on the protein and their main advantages and disadvantages. Furthermore, methods for determining protein concentration and protein storage are also presented. The guidelines compiled herein will aid preparing pure, soluble and homogeneous functional recombinant proteins from the very beginning of the molecular cloning design.

In a randomized trial, 6545 patients with acute heart failure were assigned to either serelaxin or placebo in addition to standard care. There were no significant differences between the two groups in the incidence of death from cardiovascular causes at 180 days or worsening heart failure at 5 days.

Borrelia , spirochetes transmitted by ticks, are the etiological agents of numerous multisystemic diseases, such as Lyme borreliosis (LB) and tick-borne relapsing fever (TBRF). This study focuses on two surface proteins from two Borrelia subspecies involved in these diseases: CspZ, expressed by Borrelia burgdorferi sensu stricto (also named BbCRASP-2 for complement regulator-acquiring surface protein 2), and the factor H binding A (FhbA), expressed by Borrelia hermsii. Numerous subspecies of Borrelia , including these latter, are able to evade the immune defenses of a variety of potential vertebrate hosts in a number of ways. In this context, previous data suggested that both surface proteins play a role in the immune evasion of both Borrelia subspecies by interacting with key regulators of the alternative pathway of the human complement system, factor H (FH) and FH-like protein 1 (FHL-1). The recombinant proteins, CspZ and FhbA, were expressed in Escherichia coli and purified by one-step metal-affinity chromatography, with yields of 15 and 20 mg or pure protein for 1 L of cultured bacteria, respectively. The purity was evaluated by SDS-PAGE and HPLC and is close to about 95%. The mass of CspZ and FhbA was checked by mass spectrometry (MS). Proper folding of CspZ and FhbA was confirmed by circular dichroism (CD), and their biological activity, namely their interaction with purified FH from human serum (recombinant FH 15-20  and recombinant FHL-1), was characterized by SPR. Such a study provides the basis for the biochemical characterization of the studied proteins and their biomolecular interactions which is a necessary prerequisite for the development of new approaches to improve the current diagnosis of LB and TBRF. Key points • DLS, CD, SEC-MALS, NMR, HPLC, and MS are tools for protein quality assessment • Borrelia spp. possesses immune evasion mechanisms, including human host complement • CspZ and FhbA interact with high affinity (pM to nM) to human FH and rFHL-1 Graphical Abstract

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens , is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of β-PFTs (aβ-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double β-barrel, a common feature of the aβ-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications. Epsilon toxin (Etx) is a potent pore forming toxin (PFT) produced by Clostridium perfringens. Here authors show the cryo-EM structure of the Etx pore assembled on the membrane of susceptible cells and shed light on pore formation and mutant phenotypes.

Background Escherichia coli ( E. coli ) is a popular system for recombinant protein production, owing to its low cost and availability of genetic tools. However, the expression of soluble recombinant proteins remains an issue. As such, various solubility-enhancing and yield-improving methods such as the addition of fusion tags have been developed. This study focuses on a small solubility tag (NT11), derived from the N-terminal domain of a duplicated carbonic anhydrase from Dunaliella species. The small size of NT11 (< 10 kDa) lowers the chance of protein folding interference and post-translation removal requirement, which ultimately minimizes cost of production. Results A comprehensive analysis was performed to improve the characteristics of the 11-amino acid tag. By investigating the alanine-scan library of NT11, we achieved at least a two-fold increase in protein yield for three different proteins and identified key residues for further development. We also demonstrated that the NT11 tag is not limited to the N-terminal position and can function at either the N- or C-terminal of the protein, providing flexibility in designing constructs. With these new insights, we have successfully doubled the recombinant soluble protein yields of valuable growth factors, such as fibroblast growth factor 2 (FGF2) and human epidermal growth factor (hEGF) in E. coli. Conclusion The further characterisation and development of the NT11 tag have provided valuable insights into the optimisation process for such small tags and expanded our understanding of its potential applications. The ability of the NT11 tag to be positioned at either the N- or C- termini within the protein construct without compromising its effectiveness to enhance soluble recombinant protein yields, makes it a valuable tool across a diverse range of proteins. Collectively, these findings demonstrate a promising approach to simplify and enhance the efficiency of soluble recombinant protein production.

Abstract Endolysins are bacteriophage-encoded peptidoglycan-degrading enzymes with potential applications for treating multidrug-resistant bacterial infections. While exogenously applied endolysins are active against Gram-positive bacteria in their native form, Gram-negative bacteria are protected from such activity of most native endolysins by an outer membrane. However, it was shown that recombinant endolysins can be designed to efficiently lyse Gram-negative bacteria from without as well. During our previous efforts, we purified and structurally characterized the enzymatically active domain (EAD) of phage Enc34 endolysin. In this work, we investigated the lytic potential of products resulting from different variants of fusions involving this EAD with a panel of selected antimicrobial peptides. A set of constructs was generated and expressed in Escherichia coli cells. While most such recombinant proteins accumulated intracellularly, some of them could lyse cells from within and appear in the expression medium. The fusion protein variants produced were purified and tested for their bactericidal activity against Gram-negative bacteria. The best candidate caused rapid degradation of E. coli XL1-Blue cells during the first minutes after addition, reducing the viable cell count more than three-fold. We believe that these results might be helpful in the design of new antibacterial tools. Recombinant design of the enzymatically active domain of phage Enc34 endolysin to improve its activity against Gram-negative bacteria.

Ouzounov et al . report calcium imaging with three-photon microscopy in the mouse brain. The approach enabled noninvasive recording of activity with high spatial and temporal resolution from GCaMP6-labeled neurons located as deep as the hippocampus. High-resolution optical imaging is critical to understanding brain function. We demonstrate that three-photon microscopy at 1,300-nm excitation enables functional imaging of GCaMP6s-labeled neurons beyond the depth limit of two-photon microscopy. We record spontaneous activity from up to 150 neurons in the hippocampal stratum pyramidale at ∼1-mm depth within an intact mouse brain. Our method creates opportunities for noninvasive recording of neuronal activity with high spatial and temporal resolution deep within scattering brain tissues.

Clostridium perfringens ε-toxin (ETX) is a potent pore-forming toxin responsible for a central nervous system (CNS) disease in ruminant animals with characteristics of blood-brain barrier (BBB) dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS), a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL). While native Chinese Hamster Ovary (CHO) cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/-) mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity.

The global food production system faces several challenges, including significant environmental impacts due to traditional agricultural practices. The rising demands of consumers for food products that are safe, healthy, and have animal welfare standards have led to an increased interest in alternative proteins and the development of the cellular agriculture field. Within this innovative field, precision fermentation has emerged as a promising technological solution to produce proteins with reduced ecological footprints. This review provides a summary of the environmental impacts related to the current global food production, and explores how precision fermentation can contribute to address these issues. Additionally, we report on the main animal-derived proteins produced by precision fermentation, with a particular focus on those used in the food and nutraceutical industries. The general principles of precision fermentation will be explained, including strain and bioprocess optimization. Examples of efficient recombinant protein production by bacteria and yeasts, such as milk proteins, egg-white proteins, structural and flavoring proteins, will also be addressed, along with case examples of companies producing these recombinant proteins at a commercial scale. Through these examples, we explore how precision fermentation supports sustainable food production and holds the potential for significant innovations in the sector.

Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signaling cascades, enabling them to stably maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signaling molecules (PP-InsPs), which are sensed by SPX domain-containing proteins. In plants, PP-InsP-bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP 8 –SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP 8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors. Plants regulate phosphate homeostasis via the interaction of PHR transcription factors with SPX receptors bound to inositol pyrophosphate signaling molecules. Here the authors show that inositol pyrophosphate-bound SPX interacts with the coiled-coil domain of PHR, which regulates the oligomerization and activity of the transcription factor.