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.     

Mitotic phosphorylation activates hepatoma-derived growth factor as a mitogen

Background  

Hepatoma-derived growth factor (HDGF) is a nuclear protein that is a mitogen for a wide variety of cells. Mass spectrometry based methods have identified HDGF as a phosphoprotein without validation or a functional consequence of this post-translational modification.     

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.     

RNAi-based validation of antibodies for reverse phase protein arrays

Background  

Reverse phase protein arrays (RPPA) have been demonstrated to be a useful experimental platform for quantitative protein profiling in a high-throughput format. Target protein detection relies on the readout obtained from a single detection antibody. For this reason, antibody specificity is a key factor for RPPA. RNAi allows the specific knockdown of a target protein in complex samples and was therefore examined for its utility to assess antibody performance for RPPA applications.     

Diagnostic strategies for C-reactive protein

Background  

Serum C-reactive protein (CRP) has been identified in prospective epidemiological research as an independent risk marker for cardiovascular disease. In this paper, short-term biological variation of CRP is documented and a strategy to test the reliability of a single CRP sample is proposed.     

Prediction of GTP interacting residues,dipeptides and tripeptides in a protein from its evolutionary information

Background  

Guanosine triphosphate (GTP)-binding proteins play an important role in regulation of G-protein. Thus prediction of GTP interacting residues in a protein is one of the major challenges in the field of the computational biology. In this study, an attempt has been made to develop a computational method for predicting GTP interacting residues in a protein with high accuracy (Acc), precision (Prec) and recall (Rc).     

CABYR isoforms expressed in late steps of spermiogenesis bind with AKAPs and ropporin in mouse sperm fibrous sheath

Background  

CABYR is a polymorphic calcium-binding protein of the sperm fibrous sheath (FS) which gene contains two coding regions (CR-A and CR-B) and is tyrosine as well as serine/threonine phosphorylated during in vitro sperm capacitation. Thus far, the detailed information on CABYR protein expression in mouse spermatogenesis is lacking. Moreover, because of the complexity of this polymorphic protein, there are no data on how CABYR isoforms associate and assemble into the FS.     

Spartin activates atrophin-1-interacting protein 4 (AIP4) E3 ubiquitin ligase and promotes ubiquitination of adipophilin on lipid droplets

Background  

Spartin protein is involved in degradation of epidermal growth factor receptor and turnover of lipid droplets and a lack of expression of this protein is responsible for hereditary spastic paraplegia type 20 (SPG20). Spartin is a multifunctional protein that associates with many cellular organelles, including lipid droplets. Recent studies showed that spartin interacts with E3 ubiquitin ligases that belong to the neural precursor cell-expressed developmentally downregulated gene (Nedd4) family, including atrophin-1-interacting protein 4 (AIP4/ITCH). However, the biological importance of the spartin-AIP4 interaction remains unknown.     

PIST regulates the intracellular trafficking and plasma membrane expression of Cadherin 23

Background  

The atypical cadherin protein cadherin 23 (CDH23) is crucial for proper function of retinal photoreceptors and inner ear hair cells. As we obtain more and more information about the specific roles of cadherin 23 in photoreceptors and hair cells, the regulatory mechanisms responsible for the transport of this protein to the plasma membrane are largely unknown.     

Integrating sequence and structural biology with DAS

Background  

The Distributed Annotation System (DAS) is a network protocol for exchanging biological data. It is frequently used to share annotations of genomes and protein sequence.     

MSDmotif: exploring protein sites and motifs

Background  

Protein structures have conserved features – motifs, which have a sufficient influence on the protein function. These motifs can be found in sequence as well as in 3D space. Understanding of these fragments is essential for 3D structure prediction, modelling and drug-design. The Protein Data Bank (PDB) is the source of this information however present search tools have limited 3D options to integrate protein sequence with its 3D structure.     

Identification of two proteins that interact with the Erp virulence factor from Mycobacterium tuberculosis by using the bacterial two-hybrid system

Background  

The exported repetitive protein (erp) gene encodes a secreted 36-kDa protein with a central domain containing several proline-glycine-leucine-threonine-serine (PGLTS) repeats. It has been demonstrated that erp is a virulence-associated factor since the disruption of this gene impairs the growth of Mycobacterium bovis and Mycobacterium tuberculosis in mice.     

Characterization of the internal IRES element of the zebrafish connexin55.5 reveals functional implication of the polypyrimidine tract binding protein

Background  

Connexin55.5 (Cx55.5) is a gap junction protein with horizontal cell-restricted expression in zebrafish accumulating at dendritic sites within the receptor-horizontal cell complex in form of hemichannels where light-dependent plasticity occurs. This connexin is the first example of a gap junction protein processed to form two protein isoforms from a monocistronic message by an IRES mediated process. The nuclear occurrence of a carboxy-terminal fragment of this protein provides evidence that this gap junction protein may participate in a putative cytoplasmic to nuclear signal transfer.     

Plasma proteomic profile of sulfur mustard exposed lung diseases patients using 2-dimensional gel electrophoresis

Introduction  

Sulfur mustard "bis (2-chlroethyl) sulphide" (SM) is a chemical warfare agent that remains a threat to human health. The aim of this study was to identify protein expression signature or biomarkers that reflect chronic lung damages induced by SM exposure.     

A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli

Background  

The antiphospholipid syndrome (APS), characterised by recurrent miscarriage and thrombosis, is a significant cause of morbidity and mortality. Domain I (DI) of human beta 2 glycoprotein I (β₂GPI) is thought to contain crucial antibody binding epitopes for antiphospholipid antibodies (aPL), which are critical to the pathogenesis of APS. Expressing this protein in bacteria could facilitate studies investigating how this molecule interacts with aPL.     

The anti-tumor effect of Newcastle disease virus HN protein is influenced by differential subcellular targeting

Background  

Immunotherapy is emerging as a major player in the current standard of care for aggressive cancers such as non-small cell lung cancer (NSCLC). The Newcastle disease virus with its tumor-specific replicative and oncolytic abilities is a promising immunotherapeutic candidate. A DNA vaccine expressing the major immunogenic hemagglutinin-neuraminidase (HN) protein of this virus has shown promising results as an immunotherapeutic agent.     

Follistatin-like protein 1: a serum biochemical marker reflecting the severity of joint damage in patients with osteoarthritis

Introduction  

Follistatin-like protein 1 (FSTL1) is a secreted glycoprotein that has been implicated in arthritis pathogenesis in a mouse model. The aim of this study is to detect FSTL1 expression and to further assess its potential utility as a biomarker of joint damage in osteoarthritis (OA) patients.     

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.