Expression Of Eukaryotic Proteins Research Articles

High-efficiency production of Plasmodium falciparum lactate dehydrogenase from bacteria and its functional characterization

Plasmodium falciparum is the pathogen responsible for 90 % of all malaria cases and is associated with severe complications and the highest fatality rate. Plasmodium falciparum lactate dehydrogenase (PfLDH) is a useful biomarker for the treatment and diagnosis of malaria; however, its use is limited by low production yield and functional activity. In this study, using Escherichia coli Rosetta(DE3) strain specialized for eukaryotic protein expression, we successfully expressed and purified PfLDH without a codon optimization process, which has previously been considered essential for protein expression in E. coli strains. The induction temperature and time were optimized for the overexpression of PfLDH using the transformed strain, and 31.3 mg/L of PfLDH protein was successfully purified using immobilized metal affinity chromatography. The physical properties of the purified PfLDH were assessed by verifying tetramer formation, and the functional properties of PfLDH were assessed with a colorimetric assay using a substrate that reacts with PfLDH. Subsequently, the binding of PfLDH with a DNA aptamer, which is known to specifically bind to PfLDH, was verified. These results are expected to provide important suggestions for research on obtaining large amounts of PfLDH from bacteria, thereby facilitating the treatment and diagnosis of malaria.

Engineering and characterization of GFP-targeting nanobody: Expression, purification, and post-translational modification analysis

Nanobodies are single-variable domain antibodies with excellent properties, which are evolving as versatile tools to guide cognate antigens in vitro and in vivo for biological research, diagnosis, and treatment. Given their simple structure, nanobodies are readily produced in multiple systems. However, selecting an appropriate expression system is crucial because different conditions might cause proteins to produce different folds or post-translational modifications (PTMs), and these differences often result in different functions. At present, the strategies of PTMs are rarely reported. The GFP nanobody can specifically target the GFP protein. Here, we engineered a GFP nanobody fused with 6 × His tag and Fc tag, respectively, and expressed in bacteria and mammalian cells. The 6 × His-GFP-nanobody was produced from Escherichia coli at high yields and the pull-down assay indicated that it can precipitate the GFP protein. Meanwhile, the Fc-GFP-nanobody can be expressed in HEK293T cells, and the co-immunoprecipitation experiment can trace and target the GFP-tagged protein in vivo. Furthermore, some different PTMs in antigen-binding regions have been identified after using mass spectrometry (MS) to analyze the GFP nanobodies, which are expressed in prokaryotes and eukaryotes. In this study, a GFP nanobody was designed, and its binding ability was verified by using the eukaryotic and prokaryotic protein expression systems. In addition, this GFP nanobody was transformed into a useful instrument for more in-depth functional investigations of GFP fusion proteins. MS was further used to explore the reason for the difference in binding ability, providing a novel perspective for the study of GFP nanobodies and protein expression purification.

Refolding, Crystallization, and Crystal Structure Analysis of a Scavenger Receptor Cysteine-Rich Domain of Human Salivary Agglutinin Expressed in Escherichia coli

Scavenger receptors are a protein superfamily that typically consists of one or more repeats of the scavenger receptor cysteine-rich structural domain (SRCRD), which is an ancient and highly conserved protein module. The expression and purification of eukaryotic proteins containing multiple disulfide bonds has always been challenging. The expression systems that are commonly used to express SRCRD proteins mainly consist of eukaryotic protein expression systems. Herein, we established a high-level expression strategy of a Type B SRCRD unit from human salivary agglutinin using the Escherichia coli expression system, followed by a refolding and purification process. The untagged recombinant SRCRD was expressed in E. coli using the pET-32a vector, which was followed by a refolding process using the GSH/GSSG redox system. The SRCRD expressed in E. coli SHuffle T7 showed better solubility after refolding than that expressed in E. coli BL21(DE3), suggesting the importance of the disulfide bond content prior to refolding. The quality of the refolded protein was finally assessed using crystallization and crystal structure analysis. As proteins refolded from inclusion bodies exhibit a high crystal quality and reproducibility, this method is considered a reliable strategy for SRCRD protein expression and purification. To further confirm the structural integrity of the refolded SRCRD protein, the purified protein was subjected to crystallization using sitting-drop vapor diffusion method. The obtained crystals of SRCRD diffracted X-rays to a resolution of 1.47 Å. The solved crystal structure appeared to be highly conserved, with four disulfide bonds appropriately formed. The surface charge distribution of homologous SRCRD proteins indicates that the negatively charged region at the surface is associated with their calcium-dependent ligand recognition. These results suggest that a high-quality SRCRD protein expressed by E. coli SHuffle T7 can be successfully folded and purified, providing new options for the expression of members of the scavenger receptor superfamily.

Characterization of Prolyl-4-Hydroxylase Substrate Specificity Using Pichia pastoris as an Efficient Eukaryotic Expression System.

The use of eukaryotic expression systems facilitates the heterologous expression of complex eukaryotic proteins in their post-translationally modified and biologically active state, as a prerequisite for subsequent biochemical characterization and functional analysis. Here we describe the complete workflow for the expression of Arabidopsis thaliana prolyl-4-hydroxylases (P4Hs) in the methylotrophic yeast Pichia pastoris (renamed as Komagataella phaffii), for the extraction of the recombinant enzymes, purification by affinity chromatography, and characterization of P4H activity and specificity toward oligopeptide substrates by mass spectrometry. We expressed eight of the 13 Arabidopsis P4Hs and show that they are all active against proline-rich extensin-derived peptides. However, three of them differed in substrate specificity and were also able to hydroxylate the CLEL9 signaling peptide, featuring a single proline within its mature peptide sequence.

Expression, Purification, and Characterization of Plasmodium vivax Lactate Dehydrogenase from Bacteria without Codon Optimization.

Plasmodium vivax is the most widespread cause of malaria, especially in subtropical and temperate regions such as Asia-Pacific and America. P. vivax lactate dehydrogenase (PvLDH), an essential enzyme in the glycolytic pathway, is required for the development and reproduction of the parasite. Thus, LDH from these parasites has garnered attention as a diagnostic biomarker for malaria and as a potential molecular target for developing antimalarial drugs. In this study, we prepared a transformed Escherichia coli strain for the overexpression of PvLDH without codon optimization. We introduced this recombinant plasmid DNA prepared by insertion of the PvLDH gene in the pET-21a(+) expression vector, into the Rosetta(DE3), an E. coli strain suitable for eukaryotic protein expression. The time, temperature, and inducer concentration for PvLDH expression from this E. coli Rosetta(DE3), containing the original PvLDH gene, were optimized. We obtained PvLDH with a 31.0 mg/L yield and high purity (>95%) from this Rosetta(DE3) strain. The purified protein was characterized structurally and functionally. The PvLDH expressed and purified from transformed bacteria without codon optimization was successfully demonstrated to exhibit its potential tetramer structure and enzyme activity. These findings are expected to provide valuable insights for research on infectious diseases, metabolism, diagnostics, and therapeutics for malaria caused by P. vivax.

Optimized Expression and Isolation of Recombinant Active Secreted Proteases Using Pichia pastoris.

Recombinant proteins of high quality are crucial starting materials for all downstream applications, but the inherent complexities of proteins and their expression and purification create significant challenges. The Pichia pastoris yeast is a highly useful eukaryotic protein expression system. Pichia's low cost, genetic tractability, rapid gene expression, and scalability make it an ideal expression system for foreign proteins. Here, we developed a protocol that has optimized the expression and isolation of a non-mammalian secreted metalloprotease, where we can routinely generate recombinant proteins that are pure and proteolytically active. We maximized growth and protein production by altering the feeding regime, through implementation of a non-fermentable and non-repressing carbon source during the methanol-induction phase. This approach increased biomass production and yielded milligrams of recombinant protein. Downstream applications involving active, recombinant fungal proteases, such as conjugation to nanoparticles and structure-related studies, are greatly facilitated with this improved, standardized approach. Graphical abstract.

High-Throughput Cell-Free Screening of Eukaryotic Membrane Proteins in Lipidic Mimetics.

Membrane proteins (MPs) carry out important functions in the metabolism of cells, such as the detection of extracellular activities and the transport of small molecules across the plasma and organelle membranes. Expression of MPs for biochemical, biophysical, and structural analysis is in most cases achieved by overexpression of the desired target in an appropriate host, such as a bacterium. However, overexpression of MPs is usually toxic to the host cells and can lead to aggregation of target protein and to resistance to detergent extraction. An alternative to cell-based MP expression is cell-free (CF), or in vitro, expression. CF expression of MPs has several advantages over cell-based methods, including lack of toxicity issues, no requirement for detergent extraction, and direct incorporation of target proteins in various lipidic mimetics. This article describes a high-throughput method for the expression and purification of eukaryotic membrane proteins used in the author's lab. Basic Protocol 1 describes the selection and cloning of target genes into appropriate vectors for CF expression. Basic Protocol 2 describes the assembly of CF reactions for high-throughput screening. Basic Protocol 3 outlines methods for purification and detection of target proteins. Support Protocols 1-6 describe various accessory procedures: amplification of target, treatment of vectors to prepare them for ligation-independent cloning, and the preparation of S30 extract, T7 RNA polymerase, liposomes, and nanodiscs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Target selection, construct design, and cloning into pET-based expression vectors Support Protocol 1: Amplification of target DNA Support Protocol 2: Preparation of ligation-independent cloning (LIC)-compatible vectors Basic Protocol 2: Assembly of small-scale cell-free reactions for high-throughput screening Support Protocol 3: Preparation of Escherichia coli S30 extract Support Protocol 4: Preparation of T7 RNA polymerase Support Protocol 5: Preparation of liposomes Support Protocol 6: Preparation of nanodiscs Basic Protocol 3: Purification and detection of cell-free reaction products.

Quantum Dots Tracking Endocytosis and Transport of Proteins Displayed by Mammalian Cells.

Mammalian cell display technology uses eukaryotic protein expression system to display proteins on cell surfaces and has become an important method in biological research. Although mammalian cell display technology has many advantages and development potential, certain attributes of the displayed protein remain uncharacterized, such as whether the displayed proteins re-enter the cell and how displayed proteins move into the cell. Here, we present the endocytosis mechanism, motility behavior, and transport kinetics of displayed proteins determined using HaloTag as the displayed protein and quantum dot-based single-particle tracking. The displayed protein enters the cell through clathrin-mediated endocytosis and is transported through the cell in three stages, which is dependent on microfilaments and microtubules. The dynamic information obtained in this study provides answers to questions about endocytosis and postendocytosis transport of displayed proteins and, therefore, is expected to facilitate the development of surface display technology.

Comparative transcriptome and metabolome analyses reveal the methanol dissimilation pathway of Pichia pastoris

BackgroundPichia pastoris (Komagataella phaffii) is a model organism widely used for the recombinant expression of eukaryotic proteins, and it can metabolize methanol as its sole carbon and energy source. Methanol is oxidized to formaldehyde by alcohol oxidase (AOX). In the dissimilation pathway, formaldehyde is oxidized to CO2 by formaldehyde dehydrogenase (FLD), S-hydroxymethyl glutathione hydrolase (FGH) and formate dehydrogenase (FDH).ResultsThe transcriptome and metabolome of P. pastoris were determined under methanol cultivation when its dissimilation pathway cut off. Firstly, Δfld and Δfgh were significantly different compared to the wild type (GS115), with a 60.98% and 23.66% reduction in biomass, respectively. The differential metabolites between GS115 and Δfld were mainly enriched in ABC transporters, amino acid biosynthesis, and protein digestion and absorption. Secondly, comparative transcriptome between knockout and wild type strains showed that oxidative phosphorylation, glycolysis and the TCA cycle were downregulated, while alcohol metabolism, proteasomes, autophagy and peroxisomes were upregulated. Interestingly, the down-regulation of the oxidative phosphorylation pathway was positively correlated with the gene order of dissimilation pathway knockdown. In addition, there were significant differences in amino acid metabolism and glutathione redox cycling that raised our concerns about formaldehyde sorption in cells.ConclusionsThis is the first time that integrity of dissimilation pathway analysis based on transcriptomics and metabolomics was carried out in Pichia pastoris. The blockage of dissimilation pathway significantly down-regulates the level of oxidative phosphorylation and weakens the methanol assimilation pathway to the point where deficiencies in energy supply and carbon fixation result in inefficient biomass accumulation and genetic replication. In addition, transcriptional upregulation of the proteasome and autophagy may be a stress response to resolve formaldehyde-induced DNA–protein crosslinking.

Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography.

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, as well as interactions with several growth factors and cytokines. Overexpression of specific MMPs is responsible in several diseases such as cancer, neurodegenerative diseases, and cardiovascular disease. MMPs have been the center of attention recently as targets to develop therapeutics that can treat diseases correlated to MMP overexpression. To study the MMP mechanism in solution, more facile and robust recombinant protein expression and purification methods are needed for the production of active, soluble MMPs. However, the catalytic domain of most MMPs cannot be expressed in Escherichia coli (E. coli) in soluble form due to lack of posttranslational machinery, whereas mammalian expression systems are usually costly and have lower yields. MMP inclusion bodies must undergo the tedious and laborious process of extensive purification and refolding, significantly reducing the yield of MMPs in native conformation. This paper presents a protocol using Rosetta2(DE3)pLysS (hereafter referred to as R2DP) cells to produce matrix metalloproteinase-3 catalytic domain (MMP-3cd), which contains an N-terminal His-tag followed by pro-domain (Hisx6-pro-MMP-3cd) for use in affinity purification. R2DP cells enhance the expression of eukaryotic proteins through a chloramphenicol-resistant plasmid containing codons normally rare in bacterial expression systems. Compared to the traditional cell line of choice for recombinant protein expression, BL21(DE3), purification using this new strain improved the yield of purified Hisx6-pro-MMP-3cd. Upon activation and desalting, the pro domain is cleaved along with the N-terminal His-tag, providing active MMP-3cd for immediate use in countless in vitro applications. This method does not require expensive equipment or complex fusion proteins and describes rapid production of recombinant human MMPs in bacteria.

High-throughput cell-free screening of eukaryotic membrane protein expression in lipidic mimetics.

Membrane proteins play essential roles in cellular function and metabolism. Nonetheless, biophysical and structural studies of membrane proteins are impeded by the difficulty of their expression in and purification from heterologous cell-based systems. As an alternative to these cell-based systems, cell-free protein synthesis has proven to be an exquisite method for screening membrane protein targets in a variety of lipidic mimetics. Here we report a high-throughput screening workflow and apply it to screen 61 eukaryotic membrane protein targets. For each target, we tested its expression in lipidic mimetics: two detergents, two liposomes, and two nanodiscs. We show that 35 membrane proteins (57%) can be expressed in a soluble fraction in at least one of the mimetics with the two detergents performing significantly better than nanodiscs and liposomes, in that order. Using the established cell-free workflow, we studied the production and biophysical assays for mitochondrial pyruvate carrier (MPC) complexes. Our studies show that the complexes produced in cell-free are functionally competent in complex formation and substrate binding. Our results highlight the utility of using cell-free systems for screening and production of eukaryotic membrane proteins.

Transient Transfection and Expression of Eukaryotic Membrane Proteins in Expi293F Cells and Their Screening on a Small Scale: Application for Structural Studies.

Cancers, neurodegenerative and infectious diseases remain some of the leading causes of deaths worldwide. The structure-guided drug design is essential to advance drug development for these important diseases. One of the key challenges in the structure determination workflow is the production of eukaryotic membrane proteins (drug targets) of high quality. A number of expression systems have been developed for the production of eukaryotic membrane proteins. In this chapter, an optimized detailed protocol for transient transfection and expression of eukaryotic membrane proteins in Expi293F cells is presented. Testing expression and purification on a small scale allow optimizing conditions for sample preparation for downstream structural (cryo-EM) elucidation.

Preparation of the Transient Receptor Potential Vanilloid 2 (TRPV2) channel for structural studies.

Transient Receptor Potential (TRP) channels play numerous important physiological roles in humans. Notably, they are involved in temperature sensing and regulation, in the proper functioning of immune and cardiac systems, in skin, hair, and bone physiology and in many types of cancer. Because of their physiological significance there has been much interest in elucidating their molecular mechanisms of action. Recent improvements in eukaryotic protein expression and purification techniques and in cryo-electron microscopy (cryo-EM) have greatly facilitated TRP channel studies. The TRP Vanilloid 2 (TRPV2) channel has emerged as particularly amenable to structural studies and its structure has been solved by both X-ray crystallography and by cryo-EM. Here, we provide an overview of demands posed by X-ray crystallography and cryo-EM on protein sample preparation and outline a step-by-step protocol for preparing the TRPV2 protein for structure determination by both of these techniques.

Expression and Purification of Human Mitochondrial Intramembrane Protease PARL.

Rhomboid proteases are a ubiquitous superfamily of serine intramembrane peptidases that play a role in a wide variety of cellular processes. The mammalian mitochondrial rhomboid protease, Presenilin-Associated Rhomboid Like (PARL), is a critical regulator of mitochondrial homeostasis through the cleavage of its substrates, which have roles in mitochondrial quality control and apoptosis. However, neither structural nor functional information for this important protease is available, because the expression of eukaryotic membrane proteins to sufficient levels in an active form often represents a major bottleneck for in vitro studies. Here we present an optimized protocol for expression and purification of the human PARL protease using the eukaryotic expression host Pichia pastoris. The PARL gene construct was generated in tandem with green fluorescent protein (GFP), which allowed for the selection of high expressing clones and monitoring during the large-scale expression and purification steps. We discuss the production protocol with precise details for each step. The protocol yields 1mg of pure PARL per liter of yeast culture.

Expression of recombinant multi-protein complexes in Saccharomyces cerevisiae.

Baker's yeast, Saccharomyces cerevisiae, is a versatile system for expression of recombinant eukaryotic proteins. This system is simple to use and does not require extraordinary expertise nor tissue culture facilities. Proteins expressed in the yeast system provide eukaryotic post-translational modifications, making it superior to bacterial expression for factors that require post-translational modification. In addition, it is quite simple to co-express multiple genes at the same time, for recombinant production of large multi-protein complexes. In this chapter, we provide protocols for inducible expression of recombinant genes from episomal plasmid vectors, and protocols for integration of the recombinant genes into the chromosomes of yeast, which enables simple rapid growth of expression cells and induction of recombinant protein complexes in non-selectable rich media.

Expression of eukaryotic membrane proteins in eukaryotic and prokaryotic hosts.

The production of membrane proteins of high purity and in satisfactory yields is crucial for biomedical research. Due to their involvement in various cellular processes, membrane proteins have increasingly become some of the most important drug targets in modern times. Therefore, their structural and functional characterization is a high priority. However, protein expression has always been more challenging for membrane proteins than for soluble proteins. In this review, we present four of the most commonly-used expression systems for eukaryotic membrane proteins. We describe the benefits and drawbacks of bacterial, yeast, insect and mammalian cells. In addition, we describe the different features (growth rate, yield, post-translational modifications) of each expression system, and how they are influenced by the construct design and modifications of the target gene. Cost-effective and fast-growing E. coli is mostly selected for the production of small, simple membrane proteins that, if possible, do not require post-translational modifications but has the potential for the production of bigger proteins as well. Yeast hosts are advantageous for larger and more complex proteins but for the most complex ones, insect or mammalian cells are used as they are the only hosts able to perform all the post-translational modifications found in human cells. A combination of rational construct design and host cell choice can dramatically improve membrane protein production processes.

Off-target glycans encountered along the synthetic biology route toward humanized N-glycans in Pichia pastoris.

The glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized application-customized glycan structures. In several expression hosts, this has been quite successful, but one caveat is that the new N-glycan structures inadvertently might be substrates for one or more of the multitude of endogenous glycosyltransferases in such heterologous background. This then results in the formation of novel, undesired glycan structures, which often remain insufficiently characterized. When expressing mouse interleukin-22 in a Pichia pastoris (syn. Komagataella phaffii) GlycoSwitchM5 strain, which had been optimized to produce Man5 GlcNAc2 N-glycans, glycan profiling revealed two major species: Man5 GlcNAc2 and an unexpected, partially α-mannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis, and nuclear magnetic resonance revealed that this novel glycan was Man5 GlcNAc2 modified with a Glcα-1,2-Manβ-1,2-Manβ-1,3-Glcα-1,3-R tetrasaccharide. Expression of a Golgi-targeted GlcNAc transferase-I strongly inhibited the formation of this novel modification, resulting in more homogeneous modification with the targeted GlcNAcMan5 GlcNAc2 structure. Our findings reinforce accumulating evidence that robustly customizing the N-glycosylation pathway in P. pastoris to produce particular human-type structures is still an incompletely solved synthetic biology challenge, which will require further innovation to enable safe glycoprotein pharmaceutical production.

Genetically encoded protein sulfation in mammalian cells.

Tyrosine sulfation is an important post-translational modification found in higher eukaryotes. Here we report an engineered tyrosyl-tRNA synthetase/tRNA pair that co-translationally incorporates O-sulfotyrosine in response to UAG codons in E. coli and mammalian cells. This platform enables recombinant expression of eukaryotic proteins homogeneously sulfated at chosen sites, which was demonstrated by expressing human heparin cofactor II in mammalian cells in different states of sulfation.

Ac154 carried out anti-apoptotic role during AcMNPV infection process in the host insect cells.

AcMNPV is the first baculovirus to be sequenced and is considered a model of baculovirus. ac154 is a later expression gene in AcMNPV genome and its function is unknown. Inthis study, we explored the function of Ac154 in AcMNPV infection process in host Sf9 cells. The results showed that Ac154 was distributed in both nucleus and cytoplasm. Knockout of ac154 did not affect the production of BV, but the yield of progeny virus was reduced, indicating the auxiliary function of Ac154 in virus production. MTT assay showed that Ac154 promoted the proliferation and inhibited apoptosis of Sf9 cells. Overexpression of ac154 gene significantly increased the transcription level of anti-apoptotic gene p35, and delayed the expression of the pro-apoptotic protein SfP53 and reduced its expression level, which indicated its anti-apoptotic role in the host cells. In conclusion, our results demonstrated Ac154 could delay apoptosis process in host cells by regulating the transcription of p35 gene and the expression of SfP53 protein, which provided a more favorable environment for progeny virus replication and packaging, thereby promoting the proliferation of progeny virus. So we provided a potentially improved bac-to-bac eukaryotic protein expression system and biopesticide in this work.

Improved strategy for isoleucine 1H/13C methyl labeling in Pichia pastoris

Site specific methyl labeling combined with methyl TROSY offers a powerful NMR approach to study structure and dynamics of proteins and protein complexes of high molecular weight. Robust and cost-effective methods have been developed for site specific protein 1H/13C methyl labeling in an otherwise deuterated background in bacteria. However, bacterial systems are not suitable for expression and isotope labeling of many eukaryotic and membrane proteins. The yeast Pichia pastoris (P. pastoris) is a commonly used host for expression of eukaryotic proteins, and site-specific methyl labeling of perdeuterated eukaryotic proteins has recently been achieved with this system. However, the practical utility of methyl labeling and deuteration in P. pastoris is limited by high costs. Here, we describe an improved method for 1H/13C-labeling of the δ-methyl group of isoleucine residues in a perdeuterated background, which reduces the cost by ≥ 50% without compromising the efficiency of isotope enrichment. We have successfully implemented this method to label actin and a G-protein coupled receptor. Our approach will facilitate studies of the structure and dynamics of eukaryotic proteins by NMR spectroscopy.