Crystal structure of nitrogen regulatory protein IIA<Superscript>Ntr</Superscript> from <Emphasis Type="Italic">Neisseria meningitidis</Emphasis>

Background  

The NMB0736 gene of Neisseria meningitidis serogroup B strain MC58 encodes the putative nitrogen regulatory protein, IIA^Ntr (abbreviated to NM-IIA^Ntr). The homologous protein present in Escherichia coli is implicated in the control of nitrogen assimilation. As part of a structural proteomics approach to the study of pathogenic Neisseria spp., we have selected this protein for structure determination by X-ray crystallography.     

Expression of HPV-11 L1 protein in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

Background  

We have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv. Xanthi as potential sources for an inexpensive subunit vaccine.     

Oxygen and nitrate-dependent regulation of <Emphasis Type="Italic">dmsABC</Emphasis> operon expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>: sites for Fnr and NarL protein interactions

Background  

Escherichia coli can respire anaerobically using dimethyl sulfoxide (DMSO) or trimethylamine-N-oxide (TMAO) as the terminal electron acceptor for anaerobic energy generation. Expression of the dmsABC genes that encode the membrane-associated DMSO/TMAO reductase is positively regulated during anaerobic conditions by the Fnr protein and negatively regulated by the NarL protein when nitrate is present.     

<Emphasis Type="Italic">Corynebacterium diphtheriae</Emphasis> invasion-associated protein (DIP1281) is involved in cell surface organization,adhesion and internalization in epithelial cells

Background  

Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated the function of surface-associated protein DIP1281, previously annotated as hypothetical invasion-associated protein.     

Evaluation of an <Emphasis Type="Italic">in silico</Emphasis> predicted specific and immunogenic antigen from the OmcB protein for the serodiagnosis of <Emphasis Type="Italic">Chlamydia trachomatis</Emphasis> infections

Background  

The OmcB protein is one of the most immunogenic proteins in C. trachomatis and C. pneumoniae infections. This protein is highly conserved leading to serum cross reactivity between the various chlamydial species. Since previous studies based on recombinant proteins failed to identify a species specific immune response against the OmcB protein, this study evaluated an in silico predicted specific and immunogenic antigen from the OmcB protein for the serodiagnosis of C. trachomatis infections.     

Heterologous expression of glucose oxidase in the yeast <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Background  

In spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties.     

Establishment of a Cre/loxP recombination system for N-terminal epitope tagging of genes in <Emphasis Type="Italic">Tetrahymena</Emphasis>

Background  

Epitope tagging is a powerful strategy to study the function of proteins. Although tools for C-terminal protein tagging in the ciliated protozoan Tetrahymena thermophila have been developed, N-terminal protein tagging in this organism is still technically demanding.     

Regions important for the adhesin activity of <Emphasis Type="Italic">Moraxella catarrhalis</Emphasis> Hag

Background  

The Moraxella catarrhalis Hag protein, an Oca autotransporter adhesin, has previously been shown to be important for adherence of this respiratory tract pathogen to human middle ear and A549 lung cells.     

Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Phospholipid biosynthesis commences with the acylation of glycerol-3-phosphate (G3P) to form 1-acyl-G3P. This step is catalyzed by the PlsB protein in Escherichia coli. The gene encoding this protein has not been identified, however, in the majority of bacterial genome sequences, including that of Bacillus subtilis. Recently, a new two-step pathway catalyzed by PlsX and PlsY proteins for the initiation of phospholipid formation in Streptococcus pneumoniae has been reported.     

A comparative study of protein synthesis in <Emphasis Type="Italic">in vitro</Emphasis> systems: from the prokaryotic reconstituted to the eukaryotic extract-based

Background  

Cell-free protein synthesis is not only a rapid and high throughput technology to obtain proteins from their genes, but also provides an in vitro platform to study protein translation and folding. A detailed comparison of in vitro protein synthesis in different cell-free systems may provide insights to their biological differences and guidelines for their applications.     

Tooth loss and its relationship with protein intake by elderly Brazilians—A structural equation modelling approach

Objective

This study aimed at assessing the relationship between self‐perceived tooth loss and wearing dentures, on the one hand, and the consumption of protein, on the other hand, among the elderly population of Botucatu, SP. Food consumption tends to decrease with ageing, especially protein intake, and one of the causes could be the precariousness of oral health. Several risk factors associated with deficient dietary protein intake have been identified, namely greater physical dependence, reduced caloric intake and food insecurity, but no studies have analysed whether tooth loss and prostheses interfere with protein intake.

Methods

An interview was conducted among 365 elderly individuals, in which we examined oral health‐related quality of life (OHRQoL) as the only latent variable, in a 24‐hour nutritional assessment dietary recall repeated 3 times, conducted in person by a trained nutritionist and also performed an analysis of nutritional needs using the Nutrition Data System Research (NDSR) Program.

Results

The structural equation model, performed using Stata v.14, showed that lack of teeth (standardised coefficient [SC] = 0.21, P < .001), and prosthesis use (SC = ?0.21, P < .001) was associated with OHRQoL. Lack of teeth had a direct effect on the consumption of animal protein (SC = 0.08, P = .02), a strong total effect on animal protein intake (SC = 0.51, P = .04) and a medium effect on total protein intake (SC = 0.20, P = .03), adjusted for confounders (depression and medical problems).

Conclusion

Tooth loss had a strong and significant total effect on animal protein intake and a medium effect on total protein intake among elderly Brazilians.     

Heterologous expression of a tannic acid-inducible laccase3 of <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

A tannic acid-inducible and mycoviral-regulated laccase3 (lac 3) from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast Saccharomyces cerevisiae.     

Viable nonsense mutants for the essential gene <Emphasis Type="Italic">SUP45</Emphasis> of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs) – eRF1 and eRF3. The highly conserved translation termination factor eRF1 in Saccharomyces cerevisiae is encoded by the essential gene SUP45.     

The clathrin-binding motif and the J-domain of <Emphasis Type="Italic">Drosophila Auxilin</Emphasis> are essential for facilitating Notch ligand endocytosis

Background  

Ligand endocytosis plays a critical role in regulating the activity of the Notch pathway. The Drosophila homolog of auxilin (dAux), a J-domain-containing protein best known for its role in the disassembly of clathrin coats from clathrin-coated vesicles, has recently been implicated in Notch signaling, although its exact mechanism remains poorly understood.     

Attenuation of fibrosis <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> with <Emphasis Type="Italic">SPARC</Emphasis> siRNA

Introduction  

SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs.     

Crystal structure of the YffB protein from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> suggests a glutathione-dependent thiol reductase function

Background  

The yffB (PA3664) gene of Pseudomonas aeruginosa encodes an uncharacterized protein of 13 kDa molecular weight with a marginal sequence similarity to arsenate reductase from Escherichia coli. The crystal structure determination of YffB was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein.     

<Emphasis Type="Italic">SplitTester</Emphasis> : software to identify domains responsible for functional divergence in protein family

Background  

Many protein families have undergone functional divergence after gene duplications such that current subgroups of the family carry out overlapping but distinct biological roles. For the protein families with known functional subtypes (a functional split), we developed the software, SplitTester, to identify potential regions that are responsible for the observed distinct functional subtypes within the same protein family.     

A fed‐batch based cultivation mode in Escherichia coli results in improved specific activity of a novel chimeric‐truncated form of tissue plasminogen activator

Aims

A novel chimeric‐truncated form of tissue‐type plasminogen activator (t‐PA) with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in Escherichia coli (BL21) strain and compare the protein potency in batch and fed‐batch processes.

Methods and Results

The expression cassette for the novel t‐PA was prepared in pET‐28a(+). The E. coli expression procedure was compared in traditional batch and newly developed fed batch, EnBase^® Flo system. The protein was purified in soluble format, and potency results were identified using Chromolize t‐PA Assay Kit. The fed‐batch fermentation mode, coupled with a Ni‐NTA affinity purification procedure under native condition, resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46·66 IU mg^?1) compared to traditional batch mode (35·8 IU mg^?1).

Conclusions

Considering the undeniable advantages of expression in the prokaryotic expression systems such as E. coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed‐batch cultivation methods showed the potential to replace miss‐folded formats of protein with proper folded, soluble form with improved potency.

Significance and Impact of the Study

Escherichia coli expression of recombinant proteins still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post‐translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system.