Optimization Of Protein Expression Research Articles

Experimental Study on Plasmodium knowlesi Normocyte Binding Protein Xa Region II (PkNBPXaII) for Erythrocyte Binding.

Normocyte binding protein Xa (NBPXa) has been implied to play a significant role in parasite invasion of human erythrocytes. Previous phylogenetic studies have reported the existence of three types of NBPXa for Plasmodium knowlesi (PkNBPXa). PkNBPXa region II (PkNBPXaII) of type 1, type 2 and type 3 were expressed on mammalian cell surface and interacted with human and macaque (Macaca fascicularis) erythrocytes. The binding activities of PkNBPXaII towards human and macaque erythrocytes were evaluated using erythrocyte-binding assay (EBA). Three parameters were evaluated to achieve the optimal protein expression of PkNBPXaII and erythrocyte binding activity in EBA: types of mammalian cells, post transfection time and erythrocyte incubation time. COS-7, HEK-293, and CHO-K1 cells showed successful expression of PkNBPXaII, despite the protein expression is weak compared to the positive control. COS-7 was used in EBA. All three types of PkNBPXaII showed rosette formation with macaque erythrocytes but not with human erythrocytes. Future studies to enhance the PkNBPXaII expression on surface of mammalian cells is indeed needed in order to elucidate the specific role of PkNBPXaII in erythrocytes invasion.

Expression and purification of recombinant human CUL2 from Escherichia coli cells

Cullin-2 (CUL2) based cullin RING ligases (CRL2) comprise a family of ubiquitin E3 ligases that exist only in multi-cellular organisms and are crucial for cellular processes such as embryogenesis and viral pathogenesis. In the CRL2 complex, CUL2 serves as a docking platform for the substrate receptor module, which consists of the adaptor proteins Elongin B and Elongin C (EloB/C) as well as the tumor suppressor protein VHL, where VHL is known to target hypoxia-inducible factor α (HIF1α) for ubiquitination and degradation. Abnormalities in HIF1α degradation can result in vascular tumor cell proliferation and cause a rare disease known as von-Hippel-Lindau (VHL) syndrome. Because of its critical role in HIF1α ubiquitination, CRL2VHL has been investigated for the development of novel therapeutics and treatments against disease. Given the importance of CRL2s in biological and biomedical research, a method that efficiently produces functional recombinant human CUL2 proteins will greatly facilitate studies on CRL2-dependent protein ubiquitination and degradation. Previous studies have generated recombinant human CUL2 using a baculovirus expression system. However, this method is very costly and time consuming for optimal protein expression in insect cells. To achieve a more cost-effective way of producing the human CUL2 protein, we propose to generate recombinant CUL2 in E. coli cells. We suggest that removal of unstructured regions in CUL2 would increase its solubility in E. coli. Additionally, we explored and applied the “Split-n-Co-express” strategy as previously used for CUL1 and CUL3. We report that each of our strategies resulted in soluble CUL2 expressed in E. coli cells, and following a series of purification by chromatography, the purity of the recombinant protein reached ~95%. Using in vitro assays, we found that our purified CUL2 could conjugate to NEDD8, a key activator of CRLs, and that the neddylated CUL2 could bind to VHL•EloB/C to ubiquitinate the HIF1α degron peptide. The binding and enzymatic activity of our recombinant protein demonstrate that we have established a new system for the expression and purification of functional human CUL2 from E. coli cells.

Enhanced Expression and Purification Strategy for Recombinant Bacterially-Expressed Human Hexokinase II

Dengue virus hijacks the host cellular mechanism to propagate and survive during viral infection, in which the central carbon mechanism plays a crucial role to upregulate DENV infection through the increase of human hexokinase II (HKII) activity. Since the enzyme governs the glycolytic pathway, it has potentials as a target for anti-dengue (DENV) drug development. In this study, the production of human hexokinase II protein has been enhanced by using bacterial system for anti-dengue therapeutic purpose. The HKII gene was cloned into pET28b vector and transformed into the E. coli strain BL21 (DE3) for HKII expression. In order to obtain soluble recombinant HKII in an active form, we optimized protein expression under specific conditions at 18°C for 19 hours using Terrific Broth media, in the presence of 0.5 mM isopropyl-2-D-thiogalactopyranoside (IPTG). The pET28b-HKII construct expressed in BL21(DE3) system exhibited adequate protein expression, thus, this construct was subsequently proceeded to purification process. The expressed protein was purified to homogeneity by a combination of Immobilized Metal Ion Affinity Chromatography (IMAC)and size exclusion chromatography (SEC), resulting in pure, active bacterially-expressed HKII with a specific activity of 56. 67U.mg-1. The amount of HKII obtained from 2 L culture is 80 mg, with a yield percentage of 10.5%. Hence in this study, human HKII has successfully been cloned and expressed as a soluble protein that can be utilized for further therapeutic studies.

Heterologous Expression of Interferon α-2b in Lactococcus lactis and its Biological Activity against Colorectal Cancer Cells

Lita Meilina, Sri Budiarti , Apon Zaenal Mustopa*, Huda Shalahudin Darusman, Lita Triratna, Muhammad Ajietuta Nugraha, Muhammad Sabiq Bilhaq, and Ratih Asmana Ningrum. Microbiol. Biotechnol. Lett. 2021;49:75-87. https://doi.org/10.48022/mbl.2009.09008

Optimized Adenoviral Vector That Enhances the Assembly of FMDV O1 Virus-Like Particles in situ Increases Its Potential as Vaccine for Serotype O Viruses.

Although replication-defective human adenovirus type 5 (Ad5) vectors that express in situ the capsid-encoding region of foot-and-mouth disease virus (FMDV) have been proven to be effective as vaccines in relevant species for several viral strains, the same result was not consistently achieved for the O1/Campos/Brazil/58 strain. In the present study, an optimization of the Ad5 system was explored and was proven to enhance the expression of FMDV capsid proteins and their association into virus-like particles (VLPs). Particularly, we engineered a novel Ad5 vector (Ad5[PVP2]OP) which harbors the foreign transcription unit in a leftward orientation relative to the Ad5 genome, and drives the expression of the FMDV sequences from an optimized cytomegalovirus (CMV) enhancer-promoter as well. The Ad5[PVP2]OP vaccine candidate also contains the amino acid substitutions S93F/Y98F in the VP2 protein coding sequence, predicted to stabilize FMD virus particles. Cells infected with the optimized vector showed an ∼14-fold increase in protein expression as compared to cells infected with an unmodified Ad5 vector tested in previous works. Furthermore, amino acid substitutions in VP2 protein allowed the assembly of FMDV O1/Campos/Brazil/58 VLPs. Evaluation of several serological parameters in inoculated mice with the optimized Ad5[PVP2]OP candidate revealed an enhanced vaccine performance, characterized by significant higher titers of neutralizing antibodies, as compared to our previous unmodified Ad5 vector. Moreover, 94% of the mice vaccinated with the Ad5[PVP2]OP candidate were protected from homologous challenge. These results indicate that both the optimized protein expression and the stabilization of the in situ generated VLPs improved the performance of Ad5-vectored vaccines against the FMDV O1/Campos/Brazil/58 strain and open optimistic expectations to be tested in target animals.

Development of Strong Anaerobic Fluorescent Reporters for Clostridium acetobutylicum and Clostridium ljungdahlii Using HaloTag and SNAP-tag Proteins.

One of the biggest limitations in the study and engineering of anaerobic Clostridium organisms is the lack of strong fluorescent reporters capable of strong and real-time fluorescence. Recently, we developed a strong fluorescent reporter system for Clostridium organisms based on the FAST protein. Here, we report the development of two new strong fluorescent reporter systems for Clostridium organisms based on the HaloTag and SNAP-tag proteins, which produce strong fluorescent signals when covalently bound to fluorogenic ligands. These new fluorescent reporters are orthogonal to the FAST ligands and to each other, allowing for simultaneous labeling and visualization. We used HaloTag and SNAP-tag to label the strictly anaerobic organisms Clostridium acetobutylicum and Clostridium ljungdahlii We have also identified a new strong promoter for protein expression in C. acetobutylicum, based on the phosphotransacetylase gene (pta) from C. ljungdahlii Furthermore, the HaloTag and the SNAP-tag, in combination with the previously described FAST system, were successfully used to measure cell populations in bacterial mixed cultures and showed the simultaneous orthogonal labeling of HaloTag and SNAP-tag together with the FAST protein reporter. Finally, we show the expression of recombinant fusion protein of FAST and the ZapA division protein (from C. acetobutylicum) in C. ljungdahlii. The availability of multiple strong fluorescent reporters is a major addition to the genetic toolkit of Clostridium and other anaerobes that will lead to better understanding of these unique organisms.IMPORTANCE Up to this point, assays and methods involving fluorescent reporter proteins were unavailable or limited in Clostridium organisms and other strict anaerobes. Green fluorescent protein (GFP), mCherry, and flavin-binding proteins (and their derivatives) have been used only in a few clostridia with limited success and yielded low fluorescence compared to aerobic microbial systems. Recently, we reported a new strong fluorescent reporter system based on the FAST protein as a first step in expanding the fluorescence-based reporters for Clostridium and other anaerobic microbial platforms. Additional strong orthogonal fluorescent proteins, with distinct emission spectra are needed to allow for (i) multispecies tracking within the growing field of microbial cocultures and microbiomes, (ii) protein localization and tracking in anaerobes, and (iii) identification and development of natural and synthetic promoters, ribosome-binding sites (RBS), and terminators for optimal protein expression in anaerobes. Here, we present two new strong fluorescent reporter systems based on the HaloTag and SNAP-tag proteins.

Gene design, optimization of protein expression and preliminary evaluation of a new chimeric protein for the serological diagnosis of both human and canine visceral leishmaniasis.

BackgroundVisceral leishmaniasis (VL) is a major neglected disease, potentially fatal, whose control is still impaired by inefficient and/or expensive treatment and diagnostic methods. The most promising approach for VL diagnosis uses serological assays with recombinant proteins, since they are more efficient and easier to perform. Tests developed for the human form of the disease, however, have not been shown to be efficient for its diagnosis in the canine host, the major reservoir for the American VL.Methodology/Principal findingsHere, we describe a systematic approach aimed at the production of a new chimeric protein potentially able to be used for both human and canine VL diagnosis and based both on in silico gene design and experimental data. Starting from the previous identification of Leishmania infantum recombinant antigens efficient for the diagnosis of either human or canine VL, three of the best performing antigens were selected (Lci2, Lci3 and Lci12). After a preliminary evaluation validating the chimeric approach, DNA fragments encoding predicted antigenic regions from each protein, enriched with repeats, were joined in various combinations to generate a total of seventeen chimeric genes optimized for prokaryotic expression. These were assessed for optimal expression and purification yield, with four chimeric proteins being efficiently produced. Their diagnostic potential was then evaluated through ELISA assays with sera from VL afflicted humans and dogs. After two rounds of gene design, the results showed high levels of sensitivity for the best chimeric protein, named Q5, in humans (82%) and dogs (100%) with 100% specificity in comparison with healthy controls. A single non-specific reaction was seen with serum from individuals with tegumentary leishmaniasis.ConclusionThe newly described chimeric protein is potentially useful for the detection of both humans and dogs afflicted with VL, with its use in rapid tests necessary for validation as a new diagnostic tool.

Process adapted calibration improves fluorometric pH sensor precision in sophisticated fermentation processes.

Miniaturization and automation have become increasingly popular in bioprocess development in recent years, enabling rapid high-throughput screening and optimization of process conditions. In addition, advances in the bioprocessing industry have led to increasingly complex process designs, such as pH and temperature shifts, in microbial fed-batch fermentations for optimal soluble protein expression in a range of hosts. However, in order to develop an accurate scale-down model for bioprocess screening and optimization, small-scale bioreactors must be able to accurately reproduce these complex process designs. Monitoring methods, such as fluorometric-based pH sensors, provide elegant solutions for the miniaturization of bioreactors, however, previous research suggests that the intrinsic fluorescence of biomass alters the sigmoidal calibration curve of fluorometric pH sensors, leading to inaccurate pH control. In this article, we present results investigating the impact of biomass on the accuracy of a commercially available fluorometric pH sensor. Subsequently, we present our calibration methodology for more precise online measurement and provide recommendations for improved pH control in sophisticated fermentation processes.

A bootstrap-aggregated hybrid semi-parametric modeling framework for bioprocess development.

Hybrid semi-parametric modeling, combining mechanistic and machine-learning methods, has proven to be a powerful method for process development. This paper proposes bootstrap aggregation to increase the predictive power of hybrid semi-parametric models when the process data are obtained by statistical design of experiments. A fed-batch Escherichia coli optimization problem is addressed, in which three factors (biomass growth setpoint, temperature, and biomass concentration at induction) were designed statistically to identify optimal cell growth and recombinant protein expression conditions. Synthetic data sets were generated applying three distinct design methods, namely, Box-Behnken, central composite, and Doehlert design. Bootstrap-aggregated hybrid models were developed for the three designs and compared against the respective non-aggregated versions. It is shown that bootstrap aggregation significantly decreases the prediction mean squared error of new batch experiments for all three designs. The number of (best) models to aggregate is a key calibration parameter that needs to be fine-tuned in each problem. The Doehlert design was slightly better than the other designs in the identification of the process optimum. Finally, the availability of several predictions allowed computing error bounds for the different parts of the model, which provides an additional insight into the variation of predictions within the model components.

Human 5' UTR design and variant effect prediction from a massively parallel translation assay.

The ability to predict the impact of cis-regulatory sequences on gene expression would facilitate discovery in fundamental and applied biology. Here we combine polysome profiling of a library of 280,000 randomized 5' untranslated regions (UTRs) with deep learning to build a predictive model that relates human 5' UTR sequence to translation. Together with a genetic algorithm, we use the model to engineer new 5' UTRs that accurately direct specified levels of ribosome loading, providing the ability to tune sequences for optimal protein expression. We show that the same approach can be extended to chemically modified RNA, an important feature for applications in mRNA therapeutics and synthetic biology. We test 35,212 truncated human 5' UTRs and 3,577 naturally occurring variants and show that the model predicts ribosome loading of these sequences. Finally, we provide evidence of 45 single-nucleotide variants (SNVs) associated with human diseases that substantially change ribosome loading and thus may represent a molecular basis for disease.

IRE1α-XBP1s pathway promotes prostate cancer by activating c-MYC signaling

Activation of endoplasmic reticulum (ER) stress/the unfolded protein response (UPR) has been linked to cancer, but the molecular mechanisms are poorly understood and there is a paucity of reagents to translate this for cancer therapy. Here, we report that an IRE1α RNase-specific inhibitor, MKC8866, strongly inhibits prostate cancer (PCa) tumor growth as monotherapy in multiple preclinical models in mice and shows synergistic antitumor effects with current PCa drugs. Interestingly, global transcriptomic analysis reveal that IRE1α-XBP1s pathway activity is required for c-MYC signaling, one of the most highly activated oncogenic pathways in PCa. XBP1s is necessary for optimal c-MYC mRNA and protein expression, establishing, for the first time, a direct link between UPR and oncogene activation. In addition, an XBP1-specific gene expression signature is strongly associated with PCa prognosis. Our data establish IRE1α-XBP1s signaling as a central pathway in PCa and indicate that its targeting may offer novel treatment strategies.

Optimization of Protein Expression in Mammalian Cells.

Recombinant proteins, such as monoclonal antibodies, are produced in mammalian cell lines to introduce proper protein folding and post-translational modifications, which are essential for full biological activity. In both the industrial and academic environments, the use of recombinant proteins varies widely and, with it, the method of production. The amount of an antibody needed for a toxicity study is far greater than that needed by a research lab performing cellular assays, and the amount of effort put into the development of the protein will vary accordingly. There is no universal strategy for mammalian expression systems, and scientists often struggle to develop a suitable process from the myriad of choices at each step. Here, we elaborate on the various obstacles encountered when planning high-yield experiments to produce the recombinant proteins of interest. © 2018 by John Wiley & Sons, Inc.

Determining Induction Conditions for Expression of Truncated Diphtheria Toxin and Pseudomonas Exotoxin A in E. coli BL21

Background: Targeted cancer therapies have played a great role in the treatment of malignant tumors, in the recent years. Among these therapies, targeted toxin therapies such as immunotoxins, has improved the patient’s survival rate by minimizing the adverse effect on normal tissues, whereas delivering a high dose of tumoricidal agent for eradicating the cancer tissue. Immunological proteins such as antibodies are conjugated to plant toxins or bacterial toxins such as Diphtheria toxin (DT) and Pseudomonas exotoxin A (PE) . In this case optimizing and expressing Diphtheria toxin and Pseudomonas exotoxin A which their binding domains are eliminated play a crucial role in producing the desired immunotoxins. Materials and Methods: We expressed the truncated DT and PE toxin in a genetically modified E.coli strain BL21 (DE3). For this reason we eliminated the binding domain sequences of these toxins and expressed these proteins in an expression vector pET28a with the kanamycin resistant gene for selection. The optimization of Diphtheria toxin and Pseudomonas exotoxin A expression was due to different IPTG concentration, induction and sonication time. Results: We observed that the optimal protein expression of the Diphtheria toxin was gained in 4 hours of 0.4 mM IPTG concentration at 25˚C on the other hand the optimization of Pseudomonas exotoxin A protein occurred in 4 hours of 0.5 mM IPTG concentration at 25 ˚C. Conclusion: Our study also showed lower IPTG concentrations could result in higher protein expression. By optimizing this procedure, we facilitate the protein production which could lead to acceleration of the drug development.

Accessibility of the Shine-Dalgarno Sequence Dictates N-Terminal Codon Bias in E. coli

Despite considerable efforts, no physical mechanism has been shown to explain N-terminal codon bias in prokaryotic genomes. Using a systematic study of synonymous substitutions in two endogenous E.coli genes, we show that interactions between the coding region and the upstream Shine-Dalgarno (SD) sequence modulate the efficiency of translation initiation, affecting both intracellular mRNA and protein levels due to the inherent coupling of transcription and translation in E.coli. We further demonstrate that far-downstream mutations can also modulate mRNA levels by occluding the SD sequence through the formation of non-equilibrium secondary structures. By contrast, a non-endogenous RNA polymerase that decouples transcription and translation largely alleviates the effects of synonymous substitutions on mRNA levels. Finally, a complementary statistical analysis of the E.coli genome specifically implicates avoidance of intra-molecular base pairing with the SD sequence. Our results provide general physical insights into the coding-level features that optimize protein expression in prokaryotes.

Ligand binding and activation properties of the purified bacterial cyclic nucleotide-gated channel SthK.

Cyclic nucleotide-modulated ion channels play several essential physiological roles. They are involved in signal transduction in photoreceptors and olfactory sensory neurons as well as pacemaking activity in the heart and brain. Investigations of the molecular mechanism of their actions, including structural and electrophysiological characterization, are restricted by the availability of stable, purified protein obtained from accessible systems. Here, we establish that SthK, a cyclic nucleotide-gated (CNG) channel from Spirochaeta thermophila, is an excellent model for investigating the gating of eukaryotic CNG channels at the molecular level. The channel has high sequence similarity with its eukaryotic counterparts and was previously reported to be activated by cyclic nucleotides in patch-clamp experiments with Xenopus laevis oocytes. We optimized protein expression and purification to obtain large quantities of pure, homogeneous, and active recombinant SthK protein from Escherichia coli A negative-stain electron microscopy (EM) single-particle analysis indicated that this channel is a promising candidate for structural studies with cryo-EM. Using radioactivity and fluorescence flux assays, as well as single-channel recordings in lipid bilayers, we show that the protein is partially activated by micromolar concentrations of cyclic adenosine monophosphate (cAMP) and that channel activity is increased by depolarization. Unlike previous studies, we find that cyclic guanosine monophosphate (cGMP) is also able to activate SthK, but with much lower efficiency than cAMP. The distinct sensitivities to different ligands resemble eukaryotic CNG and hyperpolarization-activated and cyclic nucleotide-modulated channels. Using a fluorescence binding assay, we show that cGMP and cAMP bind to SthK with similar apparent affinities, suggesting that the large difference in channel activation by cAMP or cGMP is caused by the efficacy with which each ligand promotes the conformational changes toward the open state. We conclude that the functional characteristics of SthK reported here will permit future studies to analyze ligand gating and discrimination in CNG channels.

An engineered E. coli ribosome with tunable translation rates enhances recombinant protein expression

Using E. coli for the expression of recombinant proteins in their soluble active form continues to be a challenge in biotechnology. Issues including low yields of full‐length, soluble, and functional proteins fundamentally limit discovery, development, and manufacturing of commercially, industrially, and medically relevant proteins. In many cases, improvements in protein solubility have been achieved by codon optimization, over‐expression of chaperone proteins, solubility tags, and by varying expression conditions. Optimization of these parameters is often done iteratively and on a case‐to‐case basis, which can be slow and expensive. To overcome limitations in recombinant protein expression, we report a genomically engineered strain of E. coli with a tunable ribosome enabling modulation in translation rates under isothermic conditions. Additionally, we show that cell extracts from this strain of E. coli can support a cell‐free protein synthesis platform capable of on‐demand production of recombinant proteins. This platform technology enables two key advances. First, we provide quantitative evidence that tuning protein synthesis rates improves soluble fractions of traditionally intractable proteins such as the human MEK1 and IRKβ. In each case, soluble fractions increased from <20% to >80% of total protein when compared with expression in BL21 cell extracts. 2) This platform technology is plug‐and‐play, allowing the user to identify the optimal translation rate condition for a desired protein in a high‐throughput manner, which can then be scaled‐up for desired protein yields. The timescale from DNA template to the optimized protein expression condition is 24–48 hours, notably faster than traditional methods involving cell transformations, and optimization of expression times and temperatures. Additionally, our data suggest that this approach obviates the need for solubility tags, enabling the expression of proteins in their wild‐type forms. Our work provides a new tool for studying protein folding in the context of ribosomal kinetics, and a new platform for manufacturing traditionally difficult proteins.Support or Funding InformationThis work was supported by DARPA and the the David and Lucille Packard Foundation Fellowship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Falcon Biomanufacturing: Teaching the core concepts of biochemistry and molecular biology through the model of a biotechnology company

To meet the challenges of the 21st century, students need a higher level of competency in science, technology, engineering, and mathematics than at any point in our history. This goes beyond terminology and abstract theory. They need to understand how different concepts relate, and most importantly how they form the basis for the technology economy. To address this need, we have developed a novel teaching platform built around the idea of a “mini‐biotech company”. The students in the biotechnology class in Bertie Early College High School, in Windsor, North Carolina learn Essential and Common Core Standards of biology and chemistry by going through the steps of making recombinant protein to be used in university research and related to the industries in their community. The year‐long program starts with a one‐week summer camp, biotech boot camp, held at NC State University. During this week, the students are introduced to concepts and techniques related to initial process development, process scale‐up, production, and validation. In addition, the students are taken to different area biotech companies where they meet working scientists and learn about their career paths. Then over the course of the next school year, the students work in their high school laboratory to develop their own process for optimal protein expression based on their equipment and available time during a school day. At the end of the school year, each student presents a poster at a biotechnology symposium held at their school, and the students that put forth the most work and leadership are selected to attend a national level science conference. Over the course of this program, students gain a deeper understanding of fundamental biochemical concepts while gaining practical, relevant work experience leading to an understanding of biotechnology beyond that of most undergraduate students.Support or Funding InformationBurroughs Wellcome Fund SSEPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nonstructural Protein σ1s Is Required for Optimal Reovirus Protein Expression.

Reovirus nonstructural protein σ1s is required for the establishment of viremia and hematogenous viral dissemination. However, the function of σ1s during the reovirus replication cycle is not known. In this study, we found that σ1s was required for efficient reovirus replication in simian virus 40 (SV40)-immortalized endothelial cells (SVECs), mouse embryonic fibroblasts, human umbilical vein endothelial cells (HUVECs), and T84 human colonic epithelial cells. In each of these cell lines, wild-type reovirus produced substantially higher viral titers than a σ1s-deficient mutant. The σ1s protein was not required for early events in reovirus infection, as evidenced by the fact that no difference in infectivity between the wild-type and σ1s-null viruses was observed. However, the wild-type virus produced markedly higher viral protein levels than the σ1s-deficient strain. The disparity in viral replication did not result from differences in viral transcription or protein stability. We further found that the σ1s protein was dispensable for cell killing and the induction of type I interferon responses. In the absence of σ1s, viral factory (VF) maturation was impaired but sufficient to support low levels of reovirus replication. Together, our results indicate that σ1s is not absolutely essential for viral protein production but rather potentiates reovirus protein expression to facilitate reovirus replication. Our findings suggest that σ1s enables hematogenous reovirus dissemination by promoting efficient viral protein synthesis, and thereby reovirus replication, in cells that are required for reovirus spread to the blood.IMPORTANCE Hematogenous dissemination is a critical step in the pathogenesis of many viruses. For reovirus, nonstructural protein σ1s is required for viral spread via the blood. However, the mechanism by which σ1s promotes reovirus dissemination is unknown. In this study, we identified σ1s as a viral mediator of reovirus protein expression. We found several cultured cell lines in which σ1s is required for efficient reovirus replication. In these cells, wild-type virus produced substantially higher levels of viral protein than a σ1s-deficient mutant. The σ1s protein was not required for viral mRNA transcription or viral protein stability. Since reduced levels of viral protein were synthesized in the absence of σ1s, the maturation of viral factories was impaired, and significantly fewer viral progeny were produced. Taken together, our findings indicate that σ1s is required for optimal reovirus protein production, and thereby viral replication, in cells required for hematogenous reovirus dissemination.

Library Growth and Protein Expression: Optimal and Reproducible Microtiter Plate Expression of Recombinant Enzymes in E. coli Using MTP Shakers.

Escherichia coli (E. coli) as heterologous host enables the recombinant expression of the desired protein in high amounts. Nevertheless, the expression in such a host, especially by utilizing a strong induction system, can result in insoluble and/or inactive protein fractions (inclusion bodies). Furthermore, the expression of different enzyme variants often leads to a diverse growth behavior of the E. coli clones resulting in the identification of false-positives when screening a mutant library. Thus, we developed a protocol for an optimal and reproducible protein expression in microtiter plates showcased for the expression of the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. By emerging this protocol, several parameters concerning the expression medium, the cultivation temperatures, shaking conditions as well as time and induction periods for CHMO were investigated. We employed a microtiter plate shaker with humidity and temperature control (Cytomat™) (integrated in a robotic platform) to obtain an even growth and expression over the plates. Our optimized protocol provides a comprehensive overview of the key factors influencing a reproducible protein expression and this should serve as basis for the adaptation to other enzyme classes.

MiPepBase: A Database of Experimentally Verified Peptides Involved in Molecular Mimicry.

Autoimmune diseases emerge due to several reasons, of which molecular mimicry i.e., similarity between the host's and pathogen's interacting peptides is an important reason. In the present study we have reported a database of only experimentally verified peptide sequences, which exhibit molecular mimicry. The database is named as miPepBase (Mimicry Peptide Database) and contains comprehensive information about mimicry proteins and peptides of both host (and model organism) and pathogen. It also provides information about physicochemical properties of protein and mimicry peptides, which might be helpful in predicting the nature of protein and optimization of protein expression. The miPepBase can be searched using a keyword or, by autoimmune disease(s) or by a combination of host and pathogen taxonomic group or their name. To facilitate the search of proteins and/or epitope in miPepBase, which is similar to the user's interest, BLAST search tool is also incorporated. miPepBase is an open access database and available at http://proteininformatics.org/mkumar/mipepbase.