Strategies to Enhance Periplasmic Recombinant Protein Production Yields in Escherichia coli

Main reasons to produce recombinant proteins in the periplasm of E. coli rather than in its cytoplasm are to -i- enable disulfide bond formation, -ii- facilitate protein isolation, -iii- control the nature of the N-terminus of the mature protein, and -iv- minimize exposure to cytoplasmic proteases. However, hampered protein targeting, translocation and folding as well as protein instability can all negatively affect periplasmic protein production yields. Strategies to enhance periplasmic protein production yields have focused on harmonizing secretory recombinant protein production rates with the capacity of the secretory apparatus by transcriptional and translational tuning, signal peptide selection and engineering, increasing the targeting, translocation and periplasmic folding capacity of the production host, preventing proteolysis, and, finally, the natural and engineered adaptation of the production host to periplasmic protein production. Here, we discuss these strategies using notable examples as a thread.

Reteplase Fc-fusions produced in N. benthamiana are able to dissolve blood clots ex vivo

Thrombolytic and fibrinolytic therapies are effective treatments to dissolve blood clots in stroke therapy. Thrombolytic drugs activate plasminogen to its cleaved form plasmin, a proteolytic enzyme that breaks the crosslinks between fibrin molecules. The FDA-approved human tissue plasminogen activator Reteplase (rPA) is a non-glycosylated protein produced in E. coli. rPA is a deletion mutant of the wild-type Alteplase that benefits from an extended plasma half-life, reduced fibrin specificity and the ability to better penetrate into blood clots. Different methods have been proposed to improve the production of rPA. Here we show for the first time the transient expression in Nicotiana benthamiana of rPA fused to the immunoglobulin fragment crystallizable (Fc) domain on an IgG1, a strategy commonly used to improve the stability of therapeutic proteins. Despite our success on the expression and purification of dimeric rPA-Fc fusions, protein instability results in high amounts of Fc-derived degradation products. We hypothesize that the “Y”- shape of dimeric Fc fusions cause steric hindrance between protein domains and leads to physical instability. Indeed, mutations of critical residues in the Fc dimerization interface allowed the expression of fully stable rPA monomeric Fc-fusions. The ability of rPA-Fc to convert plasminogen into plasmin was demonstrated by plasminogen zymography and clot lysis assay shows that rPA-Fc is able to dissolve blood clots ex vivo. Finally, we addressed concerns with the plant-specific glycosylation by modulating rPA-Fc glycosylation towards serum-like structures including α2,6-sialylated and α1,6-core fucosylated N-glycans completely devoid of plant core fucose and xylose residues.

DCMC as a Promising Alternative to Bentonite in White Wine Stabilization. Impact on Protein Stability and Wine Aromatic Fraction

Protein haze in white wine is one of the most common non-microbial defects of commercial wines, with bentonite being the main solution utilized by the winemaking industry to tackle this problem. Bentonite presents some serious disadvantages, and several alternatives have been proposed. Here, an alternative based on a new cellulose derivative (dicarboxymethyl cellulose, DCMC) is proposed. To determine the efficiency of DCMC as a bentonite alternative, three monovarietal wines were characterized, and their protein instability and content determined by a heat stability test (HST) and the Bradford method, respectively. The wines were treated with DCMC to achieve stable wines, as shown by the HST, and the efficacy of the treatments was assessed by determining, before and after treatment, the wine content in protein, phenolic compounds, sodium, calcium, and volatile organic compounds (VOCs) as well as the wine pH. DCMC applied at dosages such as those commonly employed for bentonite was able to reduce the protein content in all tested wines and to stabilize all but the Moscatel de Setúbal varietal wine. In general, DCMC was shown to induce lower changes in the wine pH and phenolic content than bentonite, reducing the wine calcium content. Regarding which VOCs are concerned, DCMC produced a general impact similar to that of bentonite, with differences depending on wine variety. The results obtained suggest that DCMC can be a sustainable alternative to bentonite in protein white wine stabilization.

Biallelic ADPRHL2 mutations in complex neuropathy affect ADP ribosylation and DNA damage response

ADP ribosylation is a reversible posttranslational modification mediated by poly(ADP-ribose)transferases (e.g., PARP1) and (ADP-ribosyl)hydrolases (e.g., ARH3 and PARG), ensuring synthesis and removal of mono-ADP-ribose or poly-ADP-ribose chains on protein substrates. Dysregulation of ADP ribosylation signaling has been associated with several neurodegenerative diseases, including Parkinson’s disease, amyotrophic lateral sclerosis, and Huntington’s disease. Recessive ADPRHL2/ARH3 mutations are described to cause a stress-induced epileptic ataxia syndrome with developmental delay and axonal neuropathy (CONDSIAS). Here, we present two families with a neuropathy predominant disorder and homozygous mutations in ADPRHL2. We characterized a novel C26F mutation, demonstrating protein instability and reduced protein function. Characterization of the recurrent V335G mutant demonstrated mild loss of expression with retained enzymatic activity. Although the V335G mutation retains its mitochondrial localization, it has altered cytosolic/nuclear localization. This minimally affects basal ADP ribosylation but results in elevated nuclear ADP ribosylation during stress, demonstrating the vital role of ADP ribosylation reversal by ARH3 in DNA damage control.

Systematic Modification and Evaluation of Enzyme-Loaded Chitosan Nanoparticles

Polymeric-based nano drug delivery systems have been widely exploited to overcome protein instability during formulation. Presently, a diverse range of polymeric agents can be used, among which polysaccharides, such as chitosan (CS), hyaluronic acid (HA) and cyclodextrins (CDs), are included. Due to its unique biological and physicochemical properties, CS is one of the most used polysaccharides for development of protein delivery systems. However, CS has been described as potentially immunogenic. By envisaging a biosafe cytocompatible and haemocompatible profile, this paper reports the systematic development of a delivery system based on CS and derived with HA and CDs to nanoencapsulate the model human phenylalanine hydroxylase (hPAH) through ionotropic gelation with tripolyphosphate (TPP), while maintaining protein stability and enzyme activity. By merging the combined set of biopolymers, we were able to effectively entrap hPAH within CS nanoparticles with improvements in hPAH stability and the maintenance of functional activity, while simultaneously achieving strict control of the formulation process. Detailed characterization of the developed nanoparticulate systems showed that the lead formulations were internalized by hepatocytes (HepG2 cell line), did not reveal cell toxicity and presented a safe haemocompatible profile.

Analysis of patient-specific NF1 variants leads to functional insights for Ras signaling that can impact personalized medicine

We have created a panel of twenty-nine NF1 variant cDNAs representing benign missense (MS) variants, pathogenic MS variants, many with clinically relevant phenotypes, in-frame deletions, splice variants, and nonsense (NS) variants. We have determined the functional consequences of the variants, assessing their ability to produce mature neurofibromin and restore Ras signaling activity in NF1 null (-/-) cells. cDNAs demonstrate variant-specific differences in neurofibromin protein levels, suggesting that some variants lead to protein instability or enhanced degradation. When expressed at high levels, some variant proteins are still able to repress Ras activity, indicating that the NF1 phenotype may be due to protein instability. In contrast, other variant proteins are incapable of repressing Ras activity, indicating that some do not functionally engage Ras and stimulate GTP-ase activity. We observed that stability and Ras activity can be mutually exclusive. These assays allow us to categorize variants by functional effects, may help to classify variants of unknown significance, and may have future implications for more directed therapeutics.

Potential Contribution of Climate Change to the Protein Haze of White Wines from the French Southwest Region

The aim of this study was to evaluate the role played by climatic conditions during grape ripening in the protein instability of white wines produced in the French southwest region. For this purpose, basic wine analyses were carried out on 268 musts and the corresponding wines, all produced during the 2016, 2017, 2018, and 2019 vintages, with distinctive climatic conditions. Qualitative and quantitative variables were correlated with levels of protein haze determined by heat test (80 °C/2 h) in the wines using analysis of covariance (ANCOVA), principal component analysis (PCA), and classification and regression trees (CART). Our results show that the climatic change, with the increase in temperatures, and the decrease in precipitation during the grape ripening phase, tends to enhance the risk of protein instability in wines. Indeed, the values of pH, titratable acidity, and malic acid concentrations of the musts, which are good indicators of the conditions in which the grapes ripened and of the level of ripeness of the grapes, were also the variables that correlated best with the protein haze. By measuring these parameters at harvest before alcoholic fermentation, it may be possible to predict the risk of protein haze, and thus early and precisely adapt the stabilization treatment to be applied.

Mutation Effect Investigation on Cytochrome C552 Protein Instability and Electron Transfer Improvement in the Acidithiobacillus Ferrooxidans Bacteria Respiratory Chain

Cytochrome c552 (Cyc1) is a protein in the electron transport chain of the Acidithiobacillus ferrooxidans (Af) bacteria which obtain their energy from oxidation Fe2+ to Fe+3. The electrons are directed through Cyc2, RCY (rusticyanin), Cytochrome c552, and Cox aa3 proteins to O2. Cytochrome c552 protein consists of two chains, A and B. In the present study, a new mutation (E121D) in the A chain of cytochrome c552 protein was selected due to electron receiving from Histidine 143 of RCY. Then, the changes performed in the E121D mutant were evaluated by MD simulations analyzes. Cytochrome c552 and RCY proteins were docked by a Patchdock server. By E121D mutation, the connection between the two chains in Cytochrome c552 was enhanced by an additional hydrogen bond between Zn1388 and aspartate 121. Asp 121 in chain A gets farther from Zn 1388 in chain B. Therefore, the aspartate gets closer to Cu 1156 of the RCY leading to the higher stability of the RCY/Cytochrome c552 complex. Further, an acidic residue (Glu121) becomes a more acidic residue (Asp121) and improving the electron transfer to Cytochrome c552 protein. The results of RMSF analysis showed further ligand flexibility in mutation. This leads to fluctuation of the active site and increases redox potential at the mutation point and the speed of electron transfer. This study also predicts that in all respiratory chain proteins, electrons probably enter the first active site via glutamate and exit through the second active site of each respiratory chain protein and through histidine.

From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes

Bioelectrocatalysis using redox enzymes appears as a sustainable way for biosensing, electricity production, or biosynthesis of fine products. Despite advances in the knowledge of parameters that drive the efficiency of enzymatic electrocatalysis, the weak stability of bioelectrodes prevents large scale development of bioelectrocatalysis. In this review, starting from the understanding of the parameters that drive protein instability, we will discuss the main strategies available to improve all enzyme stability, including use of chemicals, protein engineering and immobilization. Considering in a second step the additional requirements for use of redox enzymes, we will evaluate how far these general strategies can be applied to bioelectrocatalysis.