Predictive approaches to guide the expression of recombinant vaccine targets in Escherichia coli: a case study presentation utilising Absynth Biologics Ltd. proprietary Clostridium difficile vaccine antigens.

Bacterial expression systems remain a widely used host for recombinant protein production. However, overexpression of recombinant target proteins in bacterial systems such as Escherichia coli can result in poor solubility and the formation of insoluble aggregates. As a consequence, numerous strategies or alternative engineering approaches have been employed to increase recombinant protein production.

Esomeprazole inhibits the lysosomal cysteine protease legumain to prevent cancer metastasis.

Legumain is a newly discovered lysosomal cysteine protease that can cleave asparagine bonds and plays crucial roles in regulating immunity and cancer metastasis. Legumain has been shown to be highly expressed in various solid tumors, within the tumor microenvironment and its levels are directly related to tumor metastasis and poor prognosis. Therefore, legumain presents as a potential cancer therapeutic drug target.

Temperature Down-Shift Modifies Expression of UPR-/ERAD-Related Genes and Enhances Production of a Chimeric Fusion Protein in CHO Cells.

Low culture temperature enhances the cell-specific productivity of Chinese hamster ovary (CHO) cells expressing varied recombinant (r-) proteins, but the mechanisms remain unclear. Regulation of unfolded protein response (UPR) pathway genes, such as transcriptional regulatory factors and endoplasmic reticulum (ER)-resident proteins, appear to be involved in the improvements of r-protein production under low temperature conditions. The transcriptional regulation of UPR-specific targets is studied in response to decreased culture temperature in relation to production of a difficult-to-express protein.

Molecular Mechanism of SR Protein Kinase 1 Inhibition by the Herpes Virus Protein ICP27.

Serine-arginine (SR) protein kinase 1 (SRPK1) catalyzes the phosphorylation of SR proteins, which are a conserved family of splicing factors that contain a domain rich in arginine and serine repeats. SR proteins play important roles in constitutive pre-mRNA splicing and are also important regulators of alternative splicing. During herpes simplex virus infection, SRPK1 is inactivated and its cellular distribution is markedly altered by interaction with the viral protein ICP27, resulting in hypophosphorylation of SR proteins.

Dysregulation of macrophage development and phenotype in diabetic human macrophages can be rescued by Hoxa3 protein transduction.

Controlled inflammatory responses of myeloid cells recruited to wounds are essential for effective repair. In diabetes, the inflammatory response is prolonged and augmented over time, with increased myeloid cells present in the wound that fail to switch from a pro-inflammatory phenotype to a pro-healing phenotype. These defects lead to delayed angiogenesis and tissue repair and regeneration, and contribute to chronic wound formation.

The major secreted protein of the whipworm parasite tethers to matrix and inhibits interleukin-13 function.

Infection by soil transmitted parasitic helminths, such as Trichuris spp, are ubiquitous in humans and animals but the mechanisms determining persistence of chronic infections are poorly understood. Here we show that p43, the single most abundant protein in T. muris excretions/secretions, is non-immunogenic during infection and has an unusual sequence and structure containing subdomain homology to thrombospondin type 1 and interleukin (IL)-13 receptor (R) α2.

Surface patches on recombinant erythropoietin predict protein solubility: engineering proteins to minimise aggregation.

Background: Protein solubility characteristics are important determinants of success for recombinant proteins in relation to expression, purification, storage and administration. Escherichia coli offers a cost-efficient expression system. An important limitation, whether for biophysical studies or industrial-scale production, is the formation of insoluble protein aggregates in the cytoplasm.

Two photon spectroscopy and microscopy of the fluorescent flavoprotein, iLOV.

LOV-domains are ubiquitous photosensory proteins that are commonly re-engineered to serve as powerful and versatile fluorescent proteins and optogenetic tools. The photoactive, flavin chromophore, however, is excited using short wavelengths of light in the blue and UV regions, which have limited penetration into biological samples and can cause photodamage. Here, we have used non-linear spectroscopy and microscopy of the fluorescent protein, iLOV, to reveal that functional variants of LOV can be activated to great effect by two non-resonant photons of lower energy, near infrared light, not only in solution but also in biological samples.

Expression of recombinant proteins in insect and mammalian cells.

Purified recombinant proteins are key reagents in academic and industrial research. The ability to make these proteins quickly often relies on the availability of higher eukaryotic cell hosts such as insect and mammalian cells where there is a very wide range of post-translational modifications, protein folding and trafficking pathways. This enables the generation of many proteins that cannot be made in microbial hosts.

Functional recombinant human Legumain protein expression in Pichia pastoris to enable screening for Legumain small molecule inhibitors.

Legumain (LGMN) is a lysosomal protease that can specifically hydrolyze proteins after carboxyl-terminal asparagine residues. It has been reported that Legumain is highly expressed in many human tumors and promotes the migratory and invasive activity of cancer cells. Due to the limitation of an abundant and affordable source of endogenous active Legumain for further function studies, we produced the recombinant protein in Pichia pastoris.

PLAR binds to the annexin A2-S100A10 complex in human podocytes.

Phospholipase A receptor (PLAR) is a member of the mannose receptor family found in podocytes in human kidney. PLAR is the target of the autoimmune disease, membranous nephropathy, characterised by production of anti-PLAR autoantibodies which bind to the podocyte. However the function of PLAR in health and in disease remains unclear.

Anti-heparanase aptamers as potential diagnostic and therapeutic agents for oral cancer.

Heparanase is an endoglycosidase enzyme present in activated leucocytes, mast cells, placental tissue, neutrophils and macrophages, and is involved in tumour metastasis and tissue invasion. It presents a potential target for cancer therapies and various molecules have been developed in an attempt to inhibit the enzymatic action of heparanase. In an attempt to develop a novel therapeutic with an associated diagnostic assay, we have previously described high affinity aptamers selected against heparanase.

Identification of a major epitope recognized by PLA2R autoantibodies in primary membranous nephropathy.

Phospholipase A2 receptor 1 (PLA2R) is a target autoantigen in 70% of patients with idiopathic membranous nephropathy. We describe the location of a major epitope in the N-terminal cysteine-rich ricin domain of PLA2R that is recognized by 90% of human anti-PLA2R autoantibodies. The epitope was sensitive to reduction and SDS denaturation in the isolated ricin domain and the larger fragment containing the ricin, fibronectin type II, first and second C-type lectin domains (CTLD).

Urinary tract effects of HPSE2 mutations.

Urofacial syndrome (UFS) is an autosomal recessive congenital disease featuring grimacing and incomplete bladder emptying. Mutations of HPSE2, encoding heparanase 2, a heparanase 1 inhibitor, occur in UFS, but knowledge about the HPSE2 mutation spectrum is limited. Here, seven UFS kindreds with HPSE2 mutations are presented, including one with deleted asparagine 254, suggesting a role for this amino acid, which is conserved in vertebrate orthologs.

Heparanase induces inflammatory cell recruitment in vivo by promoting adhesion to vascular endothelium.

Heparanase (HPSE1) is known to be involved in mechanisms of metastatic tumor cell migration. This enzyme selectively cleaves heparan sulfate proteoglycans (HSPG), which are ubiquitously expressed in mammals and are known to be involved in regulating the activity of an array of inflammatory mediators. In the present study, we have investigated the effects of human recombinant heparanase, the inactive precursor of this enzyme (proheparanase) and enzymatically inactivated heparanase, on inflammatory cell recruitment in the rat and on human leukocyte-endothelial adhesion in vitro.

GAS2-like proteins mediate communication between microtubules and actin through interactions with end-binding proteins.

Crosstalk between the microtubule (MT) and actin cytoskeletons is fundamental to many cellular processes including cell polarisation and cell motility. Previous work has shown that members of the growth-arrest-specific 2 (GAS2) family mediate the crosstalk between filamentous actin (F-actin) and MTs, but the molecular basis of this process remained unclear. By using fluorescence microscopy, we demonstrate that three members of this family, GAS2-like 1, GAS2-like 2 and GAS2-like 3 (G2L1, G2L2 and G2L3, also known as GAS2L1, GAS2L2 and GAS2L3, respectively) are differentially involved in mediating the crosstalk between F-actin and MTs.

Heparanase 2, mutated in urofacial syndrome, mediates peripheral neural development in Xenopus.

Urofacial syndrome (UFS; previously Ochoa syndrome) is an autosomal recessive disease characterized by incomplete bladder emptying during micturition. This is associated with a dyssynergia in which the urethral walls contract at the same time as the detrusor smooth muscle in the body of the bladder. UFS is also characterized by an abnormal facial expression upon smiling, and bilateral weakness in the distribution of the facial nerve has been reported.

Alternative fusion protein strategies to express recalcitrant QconCAT proteins for quantitative proteomics of human drug metabolizing enzymes and transporters.

QconCAT is a tool for quantitative proteomics, consisting of an artificial protein, expressed from an artificial gene, made up of a concatenated string of proteotypic peptides selected from the proteins under study. Isotopically labeled QconCAT (usually containing (13)C6-arginine and (13)C6-lysine) provides a standard for each proteotypic peptide included in its sequence. In practice, some QconCAT proteins fail to express at sufficient levels for the purpose of quantitative analysis.

Urofacial syndrome: a genetic and congenital disease of aberrant urinary bladder innervation.

The urofacial, or Ochoa, syndrome is characterised by congenital urinary bladder dysfunction together with an abnormal grimace upon smiling, laughing and crying. It can present as fetal megacystis. Postnatal features include urinary incontinence and incomplete bladder emptying due to simultaneous detrusor muscle and bladder outlet contractions.

Anti-PLA2R antibodies measured by ELISA predict long-term outcome in a prevalent population of patients with idiopathic membranous nephropathy.

Antibodies to the phospholipase A2 receptor 1 (PLA2R1) have been reported in 70% of cases of idiopathic membranous nephropathy (IMN). The genetic susceptibility of IMN has been accounted for by HLA DQA1 and PLA2R1 genes. Here we retrospectively quantified PLA2R antibodies by ELISA, and genotyped DQ alleles and PLA2R1 single-nucleotide polymorphisms for association with clinical criteria for disease activity at the time of first sample and with outcome over a median total follow-up of 90 months.

LRIG2 mutations cause urofacial syndrome.

Urofacial syndrome (UFS) (or Ochoa syndrome) is an autosomal-recessive disease characterized by congenital urinary bladder dysfunction, associated with a significant risk of kidney failure, and an abnormal facial expression upon smiling, laughing, and crying. We report that a subset of UFS-affected individuals have biallelic mutations in LRIG2, encoding leucine-rich repeats and immunoglobulin-like domains 2, a protein implicated in neural cell signaling and tumorigenesis. Importantly, we have demonstrated that rare variants in LRIG2 might be relevant to nonsyndromic bladder disease.

Development of novel single-stranded nucleic acid aptamers against the pro-angiogenic and metastatic enzyme heparanase (HPSE1).

Heparanase is an enzyme involved in extracellular matrix remodelling and heparan sulphate proteoglycan catabolism. It is secreted by metastatic tumour cells, allowing them to penetrate the endothelial cell layer and basement membrane to invade target organs. The release of growth factors at the site of cleaved heparan sulphate chains further enhance the potential of the tumour by encouraging the process of angiogenesis.

Mutations in HPSE2 cause urofacial syndrome.

Urinary voiding dysfunction in childhood, manifesting as incontinence, dysuria, and urinary frequency, is a common condition. Urofacial syndrome (UFS) is a rare autosomal recessive disease characterized by facial grimacing when attempting to smile and failure of the urinary bladder to void completely despite a lack of anatomical bladder outflow obstruction or overt neurological damage. UFS individuals often have reflux of infected urine from the bladder to the upper renal tract, with a risk of kidney damage and renal failure.

Phosphoregulation of human Mps1 kinase.

The dual-specificity protein kinase Mps1 (monopolar spindle 1) is a phosphoprotein required for error-free mitotic progression in eukaryotes. In the present study, we have investigated human Mps1 phosphorylation using combined mass spectrometric, mutational and phosphospecific antibody approaches. We have identified 16 sites of Mps1 autophosphorylation in vitro, several of which are required for catalytic activity after expression in bacteria or in cultured human cells.