Optimization Of Protein Expression Research Articles

Noise robustness and metabolic load determine the principles of central dogma regulation.

The processes of gene expression are inherently stochastic, even for essential genes required for growth. How does the cell maximize fitness in light of noise? To answer this question, we build a mathematical model to explore the trade-off between metabolic load and growth robustness. The model provides insights for principles of central dogma regulation: Optimal protein expression levels for many genes are in vast overabundance. Essential genes are transcribed above a lower limit of one message per cell cycle. Gene expression is achieved by load balancing between transcription and translation. We present evidence that each of these regulatory principles is observed. These results reveal that robustness and metabolic load determine the global regulatory principles that govern gene expression processes, and these principles have broad implications for cellular function.

Using peptide barcodes for simultaneous profiling of protein expression from mRNA.

mRNA technology has begun to play a significant role in the areas of therapeutic intervention and vaccine development. However, optimizing the mRNA sequence that influences protein expression levels is a resource-intensive and time-consuming process. This study introduces a new method to accelerate the selection of sequences of mRNA for optimal protein expression. We designed the mRNA sequences in such a way that a unique peptide barcode, corresponding to each mRNA sequence, is attached to the expressed protein. These barcodes, cleaved off by a protease and simultaneously quantified by mass spectrometry, reflect the protein expression, enabling a parallel analysis. We validated this method using two mRNAs, each with different untranslated regions (UTRs) but encoding enhanced green fluorescence protein (eGFP), and investigated whether the peptide barcodes could analyze the differential eGFP expression levels. The fluorescence intensity of eGFP, a marker of its expression level, has shown noticeable changes between the two UTR sequences in mRNA-transfected cells when measured using flow cytometry. This suggests alterations in the expression level of eGFP due to the influence of different UTR sequences. Furthermore, the quantified amount of peptide barcodes that were released from eGFP showed consistent patterns with these changes. The experimental findings suggest that peptide barcodes serve as a valuable tool for assessing protein expression levels. The process of mRNA sequence selection, aimed at maximizing protein expression, can be enhanced by the parallel analysis of peptide barcodes using mass spectrometry.

Proteomic analysis reveals that the co-ordination of cytosolic and mitochondrial pathways is beneficial for sabinene biosynthesis in engineered Saccharomyces cerevisiae.

Reconstruction and optimization of biosynthetic pathways can help to overproduce target chemicals in microbial cell factories based on genetic engineering. However, the perturbation of biosynthetic pathways on cellular metabolism is not well investigated and profiling the engineered microbes remains challenging. The rapid development of omics tools has the potential to characterize the engineered microbial cell factory. Here, we performed label-free quantitative proteomic analysis and metabolomic analysis of engineered sabinene overproducing Saccharomyces cerevisiae strains. Combined metabolic analysis andproteomic analysis of targeted mevalonate (MVA) pathway showed that co-ordination of cytosolic and mitochondrial pathways had balanced metabolism, and genome integration of biosynthetic genes had higher sabinene production with less MVA enzymes. Furthermore, comparative proteomic analysis showed that compartmentalized mitochondria pathway had perturbation on central cellular metabolism. This study provided an omics analysis example for characterizing engineered cell factory, which can guide future regulation of the cellular metabolism and maintaining optimal protein expression levels for the synthesis of target products.

Genome-wide screen identifies new set of genes for improved heterologous laccase expression in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of the Trametes trogii fungal laccase enzyme. Gene ontology analysis of these mutants revealed an enrichment of processes including vacuolar targeting, vesicle trafficking, proteolysis, and glycolipid metabolism. We confirmed that a significant portion of these mutants also showed increased activity of the secreted laccase when grown in liquid culture. Notably, we found that the combination of deletions of OCA6, a tyrosine phosphatase gene, along with PMT1 or PMT2, two genes encoding ER membrane protein-O-mannosyltransferases involved in ER quality control, and SKI3, which encode for a component of the SKI complex responsible for mRNA degradation, further increased secreted laccase activity. Conversely, we also identified over 200 gene deletions that resulted in decreased secreted laccase activity, including many genes that encode for mitochondrial proteins and components of the ER-associated degradation pathway. Intriguingly, the deletion of the ER DNAJ co-chaperone gene SCJ1 led to almost no secreted laccase activity. When we expressed SCJ1 from a low-copy plasmid, laccase secretion was restored. However, overexpression of SCJ1 had a detrimental effect, indicating that precise dosing of key chaperone proteins is crucial for optimal recombinant protein expression. This study offers potential strategies for enhancing the overall yield of recombinant proteins and provides new avenues for further research in optimizing protein production systems.

Setup and Applications of Modular Protein Expression Toolboxes (MoPET) for Mammalian Systems.

The design and generation of an optimal protein expression construct is the first and essential step in the characterization of any protein of interest. However, the exchange and modification of the coding and/or noncoding elements to analyze their effect on protein function or generating the optimal result can be a tedious and time-consuming process using standard molecular biology cloning methods. To streamline the process to generate defined expression constructs or libraries of otherwise difficult to express proteins, the Modular Protein Expression Toolbox (MoPET) has been developed (Weber E, PloS One 12(5):e0176314, 2017). The system applies Golden Gate cloning as an assembly method and follows the standardized modular cloning (MoClo) principle (Weber E, PloS One 6(2):e16765, 2011). This cloning platform allows highly efficient DNA assembly of pre-defined, standardized functional DNA modules effecting protein expression with a focus on minimizing the cloning burden in coding regions. The original MoPET system consists of 53 defined DNA modules divided into eight functional main classes and can be flexibly expanded dependent on the need of the experimenter and expression host. However, already with a limited set of only 53 modules, 792,000 different constructs can be rationally designed or used to generate combinatorial expression optimization libraries. We provide here a detailed protocol for the (1) design and generation of level 0 basic parts, (2) generation of defined expressions constructs, and (3) generation of combinatorial expression libraries.

Chitosan Scaffolds as Microcarriers for Dynamic Culture of Human Neural Stem Cells.

Human neural stem cells (hNSCs) possess remarkable potential for regenerative medicine in the treatment of presently incurable diseases. However, a key challenge lies in producing sufficient quantities of hNSCs, which is necessary for effective treatment. Dynamic culture systems are recognized as a powerful approach to producing large quantities of hNSCs required, where microcarriers play a critical role in supporting cell expansion. Nevertheless, the currently available microcarriers have limitations, including a lack of appropriate surface chemistry to promote cell adhesion, inadequate mechanical properties to protect cells from dynamic forces, and poor suitability for mass production. Here, we present the development of three-dimensional (3D) chitosan scaffolds as microcarriers for hNSC expansion under defined conditions in bioreactors. We demonstrate that chitosan scaffolds with a concentration of 4 wt% (4CS scaffolds) exhibit desirable microstructural characteristics and mechanical properties suited for hNSC expansion. Furthermore, they could also withstand degradation in dynamic conditions. The 4CS scaffold condition yields optimal metabolic activity, cell adhesion, and protein expression, enabling sustained hNSC expansion for up to three weeks in a dynamic culture. Our study introduces an effective microcarrier approach for prolonged expansion of hNSCs, which has the potential for mass production in a three-dimensional setting.

CD4+ regulatory T cells lacking Helios and Eos

The transcriptional regulators that drive regulatory T (Treg) cell development and function remain partially understood. Helios (Ikzf2) and Eos (Ikzf4) are closely-related members of the Ikaros family of transcription factors. They are highly expressed in CD4+ Treg cells and functionally important for Treg cell biology, as mice deficient for either Helios or Eos are susceptible to autoimmune diseases. However, it remains unknown if these factors exhibit specific or partially redundant functions in Treg cells. Here we show that mice with germline deletions of both Ikzf2 and Ikzf4 are not very different from animals with single Ikzf2 or Ikzf4 deletions. Double knockout Treg cells differentiate normally, and efficiently suppress effector T cell proliferation in vitro. Both Helios and Eos are required for optimal Foxp3 protein expression. Surprisingly, Helios and Eos regulate different, largely non-overlapping, sets of genes. Only Helios is required for proper Treg cell aging, as Helios deficiency results in reduced Treg cell frequencies in the spleen of older animals. These results indicate that Helios and Eos are required for distinct aspects of Treg cell function.

One-Pot Efficient Bioconversion of Crocetin from Zeaxanthin via a Dual-Enzyme System

Saffron contains pharmacologic active compounds and a large number of flavor compounds. Both originate from carotenoids by biocatalyzed oxidation. Crocetin in saffron exhibits various attractive bioactivities and could be considered in the treatment of various diseases including cancer, coronary heart disease, and hypertension. However, powerful strategies for the efficient and sustainable source of crocetin are lacking. To address this issue, we demonstrated a one-pot approach to bioconversion of crocetin from zeaxanthin via a dual-enzyme system consisting of carotenoid cleavage dioxygenase 2 (CsCCD2-M1 (R192F/S323A)) and aldehyde dehydrogenase (SynALD) with optimized protein expression and reaction system. Meanwhile, it was observed that the byproduct 3-hydroxy-β-apo-8′-carotenoic acid (3-HACA) severely accumulated due to the imbalance in the activity of the CsCCD2-M1 and SynALD. A suitable molar ratio of 6:1 and the optimized dual-enzyme reaction conditions were developed to eliminate 3-HACA completely. Consequently, the efficient biosynthesis of crocetin (1.48 mg/L/h) and an 80.8% conversion rate of crocetin within 2 h were accomplished after scaling up the optimized system by 10 000 times. Moreover, over 34% of enzyme activity was still retained in the residual enzyme system after three times of recycling. This study not only provides a sustainable means of crocetin production in vitro but also presents a new insight into the rapid construction of one-pot synthesis of high-value compounds with complex structures.

Targeting cerebral diseases with enhanced delivery of therapeutic proteins across the blood-brain barrier

ABSTRACT Introduction Cerebral diseases have been threatening public physical and psychological health in the recent years. With the existence of the blood-brain barrier (BBB), it is particularly hard for therapeutic proteins like peptides, enzymes, antibodies, etc. to enter the central nervous system (CNS) and function in diagnosis and treatment in cerebral diseases. Fortunately, the past decade has witnessed some emerging strategies of delivering macromolecular therapeutic proteins across the BBB. Areas covered Based on the structure, functions, and substances transport mechanisms, various enhanced delivery strategies of therapeutic proteins were reviewed, categorized by molecule-mediated delivery strategies, carrier-mediated delivery strategies, and other delivery strategies. Expert opinion As for molecule-mediated delivery strategies, development of genetic engineering technology, optimization of protein expression and purification techniques, and mature of quality control systems all help to realize large-scale production of recombinant antibodies, making it possible to apply to the clinical practice. In terms of carrier-mediated delivery strategies and others, although nano-carriers/adeno-associated virus (AAV) are also promising candidates for delivering therapeutic proteins or genes across the BBB, some issues still remain to be further investigated, including safety concerns related to applied materials, large-scale production costs, quality control standards, combination therapies with auxiliary delivery strategies like focused ultrasound, etc.

Abstract 2060: Optimization of protein expression and purification of R2Bm mutant proteins

Abstract 2060: Optimization of protein expression and purification of R2Bm mutant proteins

Functional evolution of SARS-CoV-2 spike protein: Maintaining wide host spectrum and enhancing infectivity via surface charge of spike protein

The SARS-CoV-2 virus, which causes the COVID-19, is rapidly accumulating mutations to adapt to the hosts. We collected SARS-CoV-2 sequence data from the end of 2019 to January 2023 to analyze for their evolutionary features during the pandemic. We found that most of the SARS-CoV-2 genes are undergoing negative purifying selection, while the spike protein gene (S-gene) is undergoing rapid positive selection. From the original strain to the alpha, delta and omicron variant types, the Ka/Ks of the S-gene increases, while the Ka/Ks within one variant type decreases over time. During the evolution, the codon usage did not evolve towards optimal translation and protein expression. In contrast, only S-gene mutations showed a remarkable trend on accumulating more positive charges. This facilitates the infection via binding human ACE2 for cell entry and binding furin for cleavage. Such a functional evolution emphasizes the survival strategy of SARS-CoV-2, and indicated new druggable target to contain the viral infection. The nearly fully positively-charged interaction surfaces indicated that the infectivity of SARS-CoV-2 virus may approach a limit.

Codon Optimization Using a Recurrent Neural Network.

Codon optimization of a DNA sequence can significantly increase efficiency of protein expression, reducing the cost to manufacture biologic pharmaceuticals. Although directed methods based on such factors as codon usage bias and GC nucleotide content are often used to optimize protein expression, undirected optimization using machine learning could further improve the process by capitalizing on undiscovered patterns that exist within real DNA sequences. To explore this hypothesis, Chinese hamster DNA sequences were used to train a recurrent neural network (RNN) model of codon optimization. The model was used to generate optimized DNA sequence based on an input amino acid sequence for the example receptor programmed death-ligand 1 and for an example monoclonal antibody. When RNN-optimized sequences were transfected transiently or stably into Chinese hamster ovary cells, the resulting protein expression was as high or higher than that produced by DNA sequences optimized by conventional algorithms.

Abstract 2138: Creating next-generation biologics using a novel chemistry platform technology

Abstract Bright Peak Therapeutics is developing a portfolio of differentiated biotherapeutics using chemistry for applications in immuno-oncology and autoimmune diseases. Our unique chemical protein synthesis and engineering platform allows us to fine-tune cytokines and other proteins to interrogate and modulate biological functions by incorporating new functional modifications. Standard recombinant bacterial or cellular expression systems used to produce proteins are largely restricted to using canonical amino acids, which limits access to diverse modifications that can bestow additional functional properties. With chemical protein synthesis technology, canonical and non-canonical modifications including conjugation handles can be easily introduced, ultimately enabling a medicinal chemistry approach for engineering cytokine structures. Enhanced cytokines with differentiated biology developed using this approach can be further elaborated by conjugating to a diverse array of molecules. We first applied our technology platform to identify BPT-143, a rationally designed enhanced IL-2 variant currently in IND-enabling studies. BPT-143 is engineered to have enhanced binding to IL2Rβ and no binding to IL2Rα for improved efficacy and safety independent of the conjugation to a half-life extending 30 kDa PEG. The chemical synthesis technology is robust, reproducible, and scalable. We are applying our platform to enhance a number of other cytokines for use in immuno-oncology and autoimmune diseases. Additionally, our synthetically engineered cytokines can be easily conjugated to monoclonal antibodies as ‘payloads’ using a distinct chemical conjugation technology. A rapid and simple chemical process allows site-selective conjugation of our engineered cytokines to existing antibodies ‘as-is’ to generate novel immunocytokines (IC). This ‘off-the-shelf’ approach is orthogonal to recombinant fusion methods to create ICs and does not require complex recombinant protein expression optimization and lengthy cell-line development. Moreover, it allows rapid screening of cytokine payloads in a structure-activity relationship (SAR) format to identify dual-targeting ICs with precisely tailored properties to generate the desired biological effect. We have prepared a number of ICs including anti-PD-1/IL-2 ICs with various drug-antibody ratio (DAR) and conjugation sites within the antibody. We will provide an overview of the platform technology and present highlights of its application for discovery and development of designer therapeutic cytokines and ICs. Citation Format: Vijaya R. Pattabiraman, Matilde Arévalo Ruiz, Régis Boehringer, Benoit Hornsperger, Roy Meoded, Robert C. Tam, Bertolt Kreft. Creating next-generation biologics using a novel chemistry platform technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2138.

Intermittent induction of LEA peptide by lactose enhances the expression of insecticidal proteins in Bacillus thuringiensis.

Cry toxins from Bacillus thuringiensis (Bt) have been extensively applied in agriculture to substitute the use of chemical insecticides. We have previously reported the use of a coexpression system in which late embryogenesis abundant (LEA) peptides under the control of the lac promoter increase the expression of insecticidal proteins in Bt. The use of lactose to induce the expression of LEA peptides may be a desirable alternative to isopropyl β‐D‐thiogalactopyranoside, the most frequently used inducer for recombinant protein expression. In this study we investigated the use of lactose as an inducer for optimal protein expression. We observed enhanced insecticidal Cry protein expression by applying a simple technique based on intermittent induction, and then optimized concentration and the point of induction time from the 11th h to the 15th h. Our data suggest that intermittent induction of lactose might be a new technique for the enhancement of bacterial protein expression.

Cost-effective Purification of Membrane Proteins using a Dual-detergent Strategy.

Understanding the mechanisms of membrane protein function is critical for biomedical research and drug discovery as membrane proteins constitute ∼30% of the proteins encoded by the genomes of both lower and higher organisms and are targets for two-thirds of approved drugs worldwide. Significant progress has been made in engineering host expression systems for large-scale production of membrane proteins and in determining their three-dimensional high-resolution structures. Despite these efforts, the study of membrane proteins at the atomic level is challenging due to poor expression and extraction, low yields of functional protein, and the complexity and heterogeneity of source membranes. Structural and spectroscopic studies of any membrane protein require that the protein be extracted from its native membranes into a membrane-mimetic stable environment, which is often achieved by the use of detergents. Unfortunately, there is no magic detergent that can extract all membrane proteins and successful extraction often requires a thorough screen of detergents. Furthermore, membrane protein purification in general and the detergents used are very expensive, which puts a financial constraint on sophisticated membrane protein studies. To overcome this hurdle, a dual-detergent strategy has recently been developed and successfully applied to purify various classes of pure, stable, and functionally relevant membrane proteins in a cost-effective manner. This strategy uses an inexpensive detergent for solubilization of the desired protein from membranes and a second detergent during protein purification. In the Basic Protocol, we describe the dual-detergent strategy to significantly reduce the overall purification cost of a bacterial membrane protein using the magnesium ion channel MgtE as an example. Support Protocols are also provided for selecting a suitable E. coli strain for protein expression and the optimal detergent(s) for membrane protein solubilization. © 2022 Wiley Periodicals LLC. Basic Protocol: Expression, membrane solubilization, and cost-effective purification of MgtE Support Protocol 1: Selecting a suitable E. coli strain for optimal protein expression Support Protocol 2: Identification of suitable detergents for membrane protein solubilization.

Escherichia coli robustly expresses ATP synthase at growth rate-maximizing concentrations.

Fitness‐enhancing adaptations of protein expression and its regulation are an important aspect of bacterial evolution. A key question is whether evolution has led to optimal protein expression that maximizes immediate growth rate (short‐term fitness) in a robust manner (consistently across diverse conditions). Alternatively, they could display suboptimal short‐term fitness, because they cannot do better or because they instead strive for long‐term fitness maximization by, for instance, preparing for future conditions. To address this question, we focus on the ATP‐producing enzyme F1F0 H+‐ATPase, which is an abundant enzyme and ubiquitously expressed across conditions. Its expression is highly regulated and dependent on growth rate and nutrient conditions. For instance, during growth on sugars, when metabolism is overflowing acetate, glycolysis supplies most ATP, while H+‐ATPase is the main source of ATP synthesis during growth on acetate. We tested the optimality of H+‐ATPase expression in Escherichia coli across different nutrient conditions. In all tested conditions, wild‐type E. coli expresses its H+‐ATPase remarkably close (within a few per cent) to optimal concentrations that maximize immediate growth rate. This work indicates that bacteria can indeed achieve robust optimal protein expression for immediate growth‐rate maximization.

Comparison of total immunoglobulin G antibody responses to different protein fragments of Plasmodium vivax Reticulocyte binding protein 2b

BackgroundPlasmodium vivax is emerging as the dominant and prevalent species causing malaria in near-elimination settings outside of Africa. Hypnozoites, the dormant liver stage parasite of P. vivax, are undetectable to any currently available diagnostic test, yet are a major reservoir for transmission. Advances have been made to harness the naturally acquired immune response to identify recent exposure to P. vivax blood-stage parasites and, therefore, infer the presence of hypnozoites. This in-development diagnostic is currently able to detect infections within the last 9-months with 80% sensitivity and 80% specificity. Further work is required to optimize protein expression and protein constructs used for antibody detection.MethodsThe antibody response against the top performing predictor of recent infection, P. vivax reticulocyte binding protein 2b (PvRBP2b), was tested against multiple fragments of different sizes and from different expression systems. The IgG induced against the recombinant PvRBP2b fragments in P. vivax infected individuals was measured at the time of infection and in a year-long observational cohort; both conducted in Thailand.ResultsThe antibody responses to some but not all different sized fragments of PvRBP2b protein are highly correlated with each other, significantly higher 1-week post-P. vivax infection, and show potential for use as predictors of recent P. vivax infection.ConclusionsTo achieve P. vivax elimination goals, novel diagnostics are required to aid in detection of hidden parasite reservoirs. PvRBP2b was previously shown to be the top candidate for single-antigen classification of recent P. vivax exposure and here, it is concluded that several alternative recombinant PvRBP2b fragments can achieve equal sensitivity and specificity at predicting recent P. vivax exposure.

Recombinant Expression and Purification of Large Bacterial Multienzyme Assemblies for Biosynthetic Processes.

Many biosynthetic transformations have strict spatial and temporal requirements that necessitate the physical association of multiple enzymes for proper function. Here, we describe protocols for obtaining large multienzyme assemblies (>500kDa) by recombinant expression in Escherichia coli. We focus on assemblies from stand-alone enzymes joined by intermolecular forces rather than multiple catalytic domains from a single polypeptide chain. Details are given for strategies to optimize protein expression and to design a multi-affinity tag purification scheme for large multienzyme assemblies. These insights are drawn from our study of bacterial hydrocarbon biosynthesis.

Optimized Loopable Translation as a Platform for the Synthesis of Repetitive Proteins.

The expression of long proteins with repetitive amino acid sequences often presents a challenge in recombinant systems. To overcome this obstacle, we report a genetic construct that circularizes mRNA in vivo by rearranging the topology of a group I self-splicing intron from T4 bacteriophage, thereby enabling “loopable” translation. Using a fluorescence-based assay to probe the translational efficiency of circularized mRNAs, we identify several conditions that optimize protein expression from this system. Our data suggested that translation of circularized mRNAs could be limited primarily by the rate of ribosomal initiation; therefore, using a modified error-prone PCR method, we generated a library that concentrated mutations into the initiation region of circularized mRNA and discovered mutants that generated markedly higher expression levels. Combining our rational improvements with those discovered through directed evolution, we report a loopable translator that achieves protein expression levels within 1.5-fold of the levels of standard vectorial translation. In summary, our work demonstrates loopable translation as a promising platform for the creation of large peptide chains, with potential utility in the development of novel protein materials.

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.