Bacterial Expression System Research Articles

Yeast Surface Display System for Facilitated Production and Application of Phage Endolysin.

Bacteriophage endolysin is one of the potential alternatives of conventional antibiotics, but the intrinsic limitations of the bacterial expression system may undermine the comprehensive application of this therapeutic protein. To circumvent such limitations, we adopted a yeast surface display system as a novel expression platform for endolysin. Endolysin LysSA11 from staphylococcal phage SA11 was expressed and surface-displayed in Saccharomyces cerevisiae to exhibit sufficient antimicrobial activity against Staphylococcus aureus. Without any protein isolation or purification procedures, we showed that direct treatment of LysSA11-displaying yeast cells could accomplish a 5-log reduction of viable Staphylococcus aureus within 3 h. Furthermore, the surface-displayed LysSA11 exhibited superior stability over the soluble form of purified LysSA11 during 14 days of storage in a refrigerated environment. We suggest that the yeast surface display system is an efficient, stable, and straightforward platform for the production and antibacterial applications of endolysin.

Facile Synthesis and Characterization of a Novel Tamavidin-Luciferase Reporter Fusion Protein for Universal Signaling Applications.

Despite the avidin/biotin reaction being one of the most ubiquitous noncovalent immobilization and sensing strategies in scientific research, the ability to synthesize useful amounts of biotin-binding fusion constructs is hampered by poor solubility in bacterial expression systems. As such, there are few reports of successful genetic reporter fusions incorporating a biotin-binding partner. To address this, a sensitivity-enhanced, synthetically facile reporter fusion is developed to merge the bioluminescence output of Gaussia luciferase (Gluc) with the recently characterized biotin-binding ability of tamavidin 2 (TA2) for general and universal signaling applications in biological and analytical systems. This fusion construct enables direct bacterial expression of a reporter system incorporating two important functionalities in a 1:1 stoichiometric relationship that can provide detection of discrete events at low concentrations. Using a cold-shock expression system, highly concentrated construct can be obtained from standard culture volumes while retaining essentially native protein activity. To demonstrate feasibility and provide an example application, this fusion construct is then included in a standard target-bridged assay design for the sensitive detection of four miRNA targets.

HriGFP Novel Flourescent Protein: Expression and Applications.

Biosensors based on microbial cells have been developed to monitor environmental pollutants. These biosensors serve as inexpensive and convenient alternatives to the conventional lab based instrumental analysis of environmental pollutants. Small monomeric naturally occurring fluorescent proteins (fp) can be exploited by converting them as small biosensing devices for biomedical and environmental applications. Moreover, they can withstand exposure to denaturants, high temperature, and a wide pH range variation. The current study employs newly identified novel fluorescent protein HriGFP from Hydnophora rigida to detect environmental contaminants like heavy metals and organo-phosphorous (pesticide) compounds such as methyl parathion. The HriGFP was initially tested or its expression in bacterial systems (Gram positive and Gram negative) and later on for its biosensing capability in E coli (BL21DE3) for detection of heavy metals and methyl parathion was evaluated. Our results indicated the discrete and stable expression of HriGFP and a profound fluorescent quenching were observed in the presence of heavy metals (Hg, Cu, As) and methyl parathion. Structural analysis revealed heavy metal ions binding to HriGFP via amino acid residues. In-silico-analysis further revealed strong interaction via hydrogen bonds between methyl parathion phosphate oxygen atoms and the amino group of Arg119 of HriGFP. This study implies that HriGFP can act as a biosensor for detecting harmful carcinogenic pesticide like methyl parathion in water resources in the vicinity of heavily pesticide impregnated agricultural lands and heavy metal contaminated water bodies around industrial areas.

Aggregation-prone peptides modulate activity of bovine interferon gamma released from naturally occurring protein nanoparticles

Efficient protocols for the production of recombinant proteins are indispensable for the development of the biopharmaceutical sector. Accumulation of recombinant proteins in naturally-occurring protein aggregates is detrimental to biopharmaceutical development. In recent years, the view of protein aggregates has changed with the recognition that they are a valuable source of functional recombinant proteins. In this study, bovine interferon-gamma (rBoIFN-γ) was engineered to enhance the formation of protein aggregates, also known as protein nanoparticles (NPs), by the addition of aggregation-prone peptides (APPs) in the generally recognized as safe (GRAS) bacterial Lactococcus lactis expression system. The L6K2, HALRU and CYOB peptides were selected to assess their intrinsic aggregation capability to nucleate protein aggregation. These APPs enhanced the tendency of the resulting protein to aggregate at the expense of total protein yield. However, fine physico-chemical characterization of the resulting intracellular protein NPs, the protein released from them and the protein purified from the soluble cell fraction indicated that the compactability of protein conformations was directly related to the biological activity of variants of IFN-γ, used here as a model protein with therapeutic potential. APPs enhanced the aggregation tendency of fused rBoIFN-γ while increasing compactability of protein species. Biological activity of rBoIFN-γ was favored in more compacted conformations. Naturally-occurring protein aggregates can be produced in GRAS microorganisms as protein depots of releasable active protein. The addition of APPs to enhance the aggregation tendency has a positive impact in overall compactability and functionality of resulting protein conformers.

Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH

APOA5 is a low-abundance exchangeable apolipoprotein that plays critical roles in human triglyceride (TG) metabolism. Indeed, aberrations in the plasma concentration or structure of APOA5 are linked to hypertriglyceridemia, hyperchylomicronemia, myocardial infarction risk, obesity, and coronary artery disease. While it has been successfully produced at low yield in bacteria, the resulting protein had limitations for structure-function studies due to its low solubility under physiological buffer conditions. We hypothesized that the yield and solubility of recombinant APOA5 could be increased by: i) engineering a fusion protein construct in a codon optimized expression vector, ii) optimizing an efficient refolding protocol, and iii) screening buffer systems at physiological pH. The result was a high-yield (25 mg/l) bacterial expression system that produces lipid-free APOA5 soluble at concentrations of up to 10 mg/ml at a pH of 7.8 in bicarbonate buffers. Physical characterization of lipid-free APOA5 indicated that it exists as an array of multimers in solution, and far UV circular dichroism analyses show differences in total α-helicity between acidic and neutral pH buffering conditions. The protein was functional in that it bound and emulsified multilamellar dimyristoyl-phosphatidylcholine vesicles and could inhibit postprandial plasma TG accumulation when injected into C57BL/6J mice orally gavaged with Intralipid.

Innate immunemodulator containing adjuvant formulated HA based vaccine protects mice from lethal infection of highly pathogenic avian influenza H5N1 virus

The highly pathogenic avian influenza (HPAI) H5N1 viruses and their spillover into the human population pose substantial economic and public health threats. Although antiviral drugs have some effect in treating influenza infection, vaccination is still the most effective intervention to prevent possible pandemic outbreaks. We have developed a novel H5 influenza vaccine to improve the world’s pandemic preparedness. We produced a hemagglutinin (HA) of HPAI H5N1 virus A/Alberta/01/2014 (AB14) using both mammalian (m) and bacterial (b) expression systems. The purified recombinant proteins were formulated with a proprietary adjuvant (TriAdj) and their efficacy as vaccine candidates was evaluated in mice. Intramuscular delivery of two doses of TriAdj formulated mammalian expressed HA (m-HA/TriAdj) was shown to provide full protection against a lethal challenge of AB14 in mice. In contrast, bacterially expressed HA with TriAdj (b-HA/TriAdj), b-HA without adjuvant, and m-HA without adjuvant resulted in no protection in immunized mice. Furthermore, m-HA/TriAdj elicited significantly higher levels of balanced Th1 and Th2 responses and neutralizing antibody titres. All the mice in the m-HA/TriAdj group survived a lethal AB14 H5N1 challenge and showed no signs of disease or infection as demonstrated by no loss of body weight or detectable virus in the lungs. Our results suggest that m-HA formulated with TriAdj has potential to protect against pandemic H5N1 in the event of its cross over to the human host.

A single-component light sensor system allows highly tunable and direct activation of gene expression in bacterial cells.

Light-regulated modules offer unprecedented new ways to control cellular behaviour with precise spatial and temporal resolution. Among a variety of bacterial light-switchable gene expression systems, single-component systems consisting of single transcription factors would be more useful due to the advantages of speed, simplicity, and versatility. In the present study, we developed a single-component light-activated bacterial gene expression system (eLightOn) based on a novel LOV domain from Rhodobacter sphaeroides (RsLOV). The eLightOn system showed significant improvements over the existing single-component bacterial light-activated expression systems, with benefits including a high ON/OFF ratio of >500-fold, a high activation level, fast activation kinetics, and/or good adaptability. Additionally, the induction characteristics, including regulatory windows, activation kinetics and light sensitivities, were highly tunable by altering the expression level of LexRO. We demonstrated the usefulness of the eLightOn system in regulating cell division and swimming by controlling the expression of the FtsZ and CheZ genes, respectively, as well as constructing synthetic Boolean logic gates using light and arabinose as the two inputs. Taken together, our data indicate that the eLightOn system is a robust and highly tunable tool for quantitative and spatiotemporal control of bacterial gene expression.

Deciphering the molecular mechanism of FLT3 resistance mutations.

FMS-like tyrosine kinase 3 (FLT3) has been found to be mutated in ~30% of acute myeloid leukaemia patients. Small-molecule inhibitors targeting FLT3 that are currently approved or still undergoing clinical trials are subject to drug resistance due to FLT3 mutations. How these mutations lead to drug resistance is hitherto poorly understood. Herein, we studied the molecular mechanism of the drug resistance mutations D835N, Y842S and M664I, which confer resistance against the most advanced inhibitors, quizartinib and PLX3397 (pexidartinib), using enzyme kinetics and computer simulations. Invitro kinase assays were performed to measure the comparative catalytic activity of the native protein and the mutants, using a bacterial expression system developed to this aim. Our results reveal that the differential drug sensitivity profiles can be rationalised by the dynamics of the protein-drug interactions and perturbation of the intraprotein contacts upon mutations. Drug binding induced a single conformation in the native protein, whereas multiple conformations were observed otherwise (in the mutants or in the absence of drugs). The end-point kinetics measurements indicated that the three resistant mutants conferred catalytic activity that is at least as high as that of the reference without such mutations. Overall, our calculations and measurements suggest that the structural dynamics of the drug-resistant mutants that affect the active state and the increased conformational freedom of the remaining inactive drug-bound population are the two major factors that contribute to drug resistance in FLT3 harbouring cancer cells. Our results explain the mechanism of drug resistance mutations and can aid to the design of more effective tyrosine kinase inhibitors.

Bacterial Expression Systems for Enzymatic Activity in Droplet-Based Microfluidics.

Functional screenings in droplet-based microfluidics require the analysis of various types of activities of individual cells. When screening for enzymatic activities, the link between the enzyme of interest and the information-baring molecule, the DNA, must be maintained to relate phenotypes to genotypes. This linkage is crucial in directed evolution experiments or for the screening of natural diversity. Micro-organisms are classically used to express enzymes from nucleic acid sequences. However, little information is available regarding the most suitable expression system for the sensitive detection of enzymatic activity at the single-cell level in droplet-based microfluidics. Here, we compare three different expression systems for l-asparaginase (l-asparagine amidohydrolase, EC 3.5.1.1), an enzyme of therapeutic interest that catalyzes the conversion of l-asparagine to l-aspartic acid and ammonia. We developed three expression vectors to produce and localize l-asparaginase (l-ASNase) in E. coli either in the cytoplasm, on the surface of the inner membrane (display), or in the periplasm. We show that the periplasmic expression is the most optimal strategy combining both a good yield and a good accessibility for the substrate without the need for lysing the cells. We suggest that periplasmic expression may provide a very efficient platform for screening applications at the single-cell level in microfluidics.

Purification of the Dynamin-Related Protein Vps1 Using Mammalian and Bacterial Expression Systems.

The dynamin-related proteins (DRPs) are self-assembling membrane remodeling machines that are indispensable for fundamental cellular trafficking and homeostatic processes. We describe in this chapter protocols developed in our laboratory for purification of full-length and minimal constructs of Chaetomium thermophilum Vps1, the model fungal DRP, using mammalian and Escherichia coli expression systems.

Bacterial expression and purification of vertebrate odorant-binding proteins.

Vertebrate odorant-binding proteins (OBPs) are small soluble proteins abundantly secreted in the olfactory mucus of many animal species, including humans. Vertebrate OBPs reversibly bind odorant molecules with micromolar range affinities. Although their physiological role is not clearly understood, OBPs are proposed to carry airborne odorants toward membrane olfactory receptors through the nasal mucus. Measurements of odorant-OBP interactions and structural studies require a large amount of pure OBPs devoid of ligands. The bacterial expression system is the first choice for expressing vertebrate OBPs used in our laboratory and others. This system generally produces OBPs in large amounts without major problems. In this chapter, we describe the milligram-scale production of recombinant pig OBP1 (pOBP1) in E. coli. The different steps of expression and purification are presented and discussed. Protocols for secondary structures investigation by circular dichroism and binding properties of the recombinant protein are also provided. More generally, these approaches can be used to produce and characterize any vertebrate OBPs for use in functional and structural studies.

Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

Recombinant Expression of Cyclotides Using Expressed Protein Ligation.

Cyclotides are naturally occurring microproteins (≈30 residues long) present in several families of plants. All cyclotides share a unique head-to-tail circular knotted topology containing three disulfide bridges forming a cystine knot topology. Cyclotides possess high stability to chemical, physical, and biological degradation and have been reported to cross cellular membranes. In addition, naturally occurring and engineered cyclotides have shown to possess various pharmacologically relevant activities. These unique features make the cyclotide scaffold an excellent tool for the design of novel peptide-based therapeutics by using molecular evolution and/or peptide epitope grafting techniques. In this chapter, we provide protocols to recombinantly produce a natively folded cyclotide making use of a standard bacterial expression system in combination with an intein-mediated backbone cyclization with concomitant oxidative folding.

In silico Analysis, Molecular Docking, Molecular Dynamic, Cloning, Expression and Purification of Chimeric Protein in Colorectal Cancer Treatment.

IntroductionColorectal cancer (CRC) is a type of cancer in humans that leads to high mortality and morbidity. CD166 and CD326 are immunoglobulins that are associated with cell migration. These molecules are included in tumorigenesis of CRC and serve a great marker of CRC stem cells. In the present study, we devised a novel chimeric protein including the V1-domain of the CD166 and two epitopes of CD326 to use in diagnostic or therapeutic applications.MethodsIn silico techniques were launched to characterize the properties and structure of the protein. We have predicted physicochemical properties, structures, stability, MHC class I binding properties and ligand-receptor interaction of this chimeric protein by means of computational bioinformatics tools and servers. The sequence of chimeric gene was optimized for expression in prokaryotic host using online tools and cloned into pET-28a plasmid. The recombinant pET28a was transformed into the E. coli BL21DE3. Expression of recombinant protein was examined by SDS-PAGE and Western blotting.ResultsThe designed chimeric protein retained high stability and the same immunogenicity as of the original proteins. Bioinformatics data indicated that the epitopes of the synthetic chimeric protein might induce B-cell- and T-cell-mediated immune responses. Furthermore, a gene was synthesized using the codon bias of a prokaryotic expression system. This synthetic gene expressed a bacterial expression system. The recombinant protein with molecular weights of 27kDa was expressed and confirmed by anti-his Western blot analysis.ConclusionThe designed recombinant protein may be useful as a CRC diagnostic tool and for developing a protective vaccine against CRC.

Screening and Identification of Novel cGAS Homologues Using a Combination of in Vitro and In Vivo Protein Synthesis

The cyclic GMP-AMP synthase (cGAS) catalyzes the synthesis of the multifunctional second messenger, cGAMP, in metazoans. Although numerous cGAS homologues are predicted in protein databases, the catalytic activity towards cGAMP synthesis has been proven for only four of them. Therefore, we selected five novel and yet uncharacterized cGAS homologues, which cover a broad range in the field of vertebrates. Cell-free protein synthesis (CFPS) was used for a pre-screening to investigate if the cGAS genes originating from higher organisms can be efficiently expressed in a bacterial expression system. As all tested cGAS variants were expressible, enzymes were synthesized in vivo to supply higher amounts for a subsequent in vitro activity assay. The assays were carried out with purified enzymes and revealed vast differences in the activity of the homologues. For the first time, the cGAS homologues from the Przewalski’s horse, naked mole-rat, bald eagle, and zebrafish were proven to catalyze the synthesis of cGAMP. The extension of the list of described cGAS variants enables the acquisition of further knowledge about the structural and molecular mechanism of cGAS, potentially leading to functional improvement of the enzyme.

Characterization, cloning, expression and bioassay of vip3 gene isolated from an Egyptian Bacillus thuringiensis against whiteflies.

Throughout the vegetative life of Bacillus thuringiensis, vegetative insecticidal proteins (Vip) are produced and secreted. In the present study, the vip3 gene isolated from Bacillus thuringiensis, an Egyptian isolate, was successfully amplified (2.4 kbp) and expressed using bacterial expression system. The molecular mass of the expressed protein was verified using SDS-PAGE and western blot analysis. Whiteflies were also screened for susceptibility to the expressed Vip3 protein (LC50). In addition, ST50 was determined to assess the kill speed of the expressed Vip3 protein against whiteflies compared to the whole vegetative proteins. The results showed that the potency of whole B. thuringiensis vegetative proteins against whiteflies was slightly higher than the expressed Vip3 protein with 4.7-fold based on LC50 value. However, the ST50 parameter showed no significant difference between both the B. thuringiensis vegetative proteins and the expressed Vip3 alone. The results showed that the vip3 gene was successfully expressed in an active form which showed high susceptibility to whiteflies based on the virulence parameters LC50 and ST50. To our knowledge, this study showed for the first time the high toxicity of the expressed Vip3 proteins of B. thuringiensis toward whiteflies as a hopeful and promising bio-control agent.

Functional interaction of bacterial virulence factors of Xenorhabdus nematophila with a calcium-independent cytosolic PLA2 of Spodoptera exigua

Phospholipase A2 (PLA2) hydrolyzes the ester bond of phospholipids (PLs) at sn-2 and releases free fatty acids and lysophospholipids that are subsequently changed into various signal molecules to mediate various physiological processes. Numerous PLA2s are known in various biological systems and can be divided into at least 16 groups. Although different PLA2s recently have been annotated from several insect species, physiological roles are known for only a few genes. Two calcium-independent PLA2s (Se-iPLA2A and Se-iPLA2B) are known in the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae). We generated and purified a recombinant Se-iPLA2B (rSe-iPLA2B) using a bacterial expression system and analyzed the enzyme kinetics. rSe-iPLA2B exhibited catalytic activities against both arachidonyl (AA)-PL and non-AA-PL substrates. It was highly susceptible to iPLA2-specific inhibitor, but insensitive to inhibitors specific to secretory PLA2s or calcium-dependent cytosolic PLA2s. Increasing calcium concentrations prevented enzyme activity, and culture medium of an entomopathogenic bacterium, Xenorhabdus nematophila, or its organic extracts significantly inhibited enzyme activity. Binding assays of rSe-iPLA2B with known secondary metabolites identified from X. nematophila indicated that benzylideneacetone was the most potent inhibitor with a high binding affinity at 0.2 μM against rSe-iPLA2B. Furthermore, rSe-iPLA2B catalyzed the release of fatty acids from PLs extracted from S. exigua fat body, suggesting its physiological role in maintaining PL integrity. All these catalytic activities indicate that Se-iPLA2B has the typical biochemical properties of other iPLA2s. Its high binding affinity to secondary metabolites of X. nematophila suggests that it is a molecular target of X. nematophila, an entomopathogen.

Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers.

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and lipids during endocytosis, sorting within the endosomal system, and autophagy. Central to SNX-BAR protein function is the ability to form homodimers or heterodimers that bind membranes using highly conserved phox-homology (PX) and BAR (Bin/Amphiphysin/Rvs) domains. In addition, oligomerization of SNX-BAR dimers on membranes can elicit the formation of membrane tubules and vesicles and this activity is thought to reflect their functions as coat proteins for endosome-derived transport carriers. Researchers have long utilized in vitro binding studies using recombinant SNX-BAR proteins on synthetic liposomes or giant unilamellar vesicles (GUVs) to reveal the precise makeup of lipids needed to drive membrane remodeling, thus revealing their mechanism of action. However, due to technical challenges with dual expression systems, toxicity of SNX-BAR protein expression in bacteria, and poor solubility of individual SNX-BAR proteins, most studies to date have examined SNX-BAR homodimers, including non-physiological dimers that form during expression in bacteria. Recently, we have optimized a protocol to overcome the major shortcomings of a typical bacterial expression system. Using this workflow, we demonstrate how to successfully express and purify large amounts of SNX-BAR heterodimers and how to reconstitute them on synthetic liposomes for binding and tubulation assays.

Non-Catalytic Subunits Facilitate Quaternary Organization of Plastidic Acetyl-CoA Carboxylase.

Arabidopsis (Arabidopsis thaliana), like most dicotyledonous plants, expresses a multicomponent, heteromeric acetyl-CoA carboxylase (htACCase), which catalyzes the generation of the malonyl-CoA precursor of de novo fatty acid biosynthesis. This enzyme consists of four catalytic subunits: biotin carboxylase (BC), carboxyltransferase (CT)-α, CT-β, and biotin carboxyl carrier protein (BCCP1 or BCCP2). By coexpressing combinations of components in a bacterial expression system, we demonstrate noncatalytic BADCs facilitate the assembly and activation of BCCP-BADC-BC subcomplexes catalyzing the bicarbonate-dependent hydrolysis of ATP, which is the first half-reaction catalyzed by the htACCase enzyme. Although BADC proteins do not directly impact formation of the CT-αβ subcomplex, the BADC-facilitated BCCP-BADC-BC subcomplex can more readily interact with the CT-αβ subcomplex to facilitate the generation of malonyl-CoA. The Arabidopsis genome encodes three BADC isoforms (BADC1, BADC2, and BADC3), and BADC2 and BADC3 (rather than BADC1), in combination with BCCP1, best support this quaternary-structural organization and catalytic activation of the htACCase enzyme. Physiological genetic studies validate these attributes as Arabidopsis double mutants singularly expressing BADC2, BADC3, or BADC1 present increasingly greater deleterious impacts on morphological and biochemical phenotypes. Specifically, plants expressing only BADC2 develop normally, plants only expressing BADC3 suffer a stunted root-growth phenotype, and plants expressing only BADC1 are embryo-lethal. The latter phenotype may also be associated with the distinct suborganelle localization of BADC1 in plastids as compared to the localization of the other two BADC homologs. These finding can inspire novel strategies to improve the biological sources of fats and oils for dietary and industrial applications.

Bacterial overexpression and purification of soluble recombinant human serum albumin using maltose-binding protein and protein disulphide isomerase

Human serum albumin (HSA), the most abundant serum protein in healthy humans, plays important roles in many physiological processes and has wide clinical and research applications. Despite several efforts to obtain recombinant HSA (rHSA) from bacterial and eukaryotic expression systems, a low-cost and high-yield method for rHSA production is not available. The large molecular weight and high disulphide content hamper the expression and production of rHSA using bacterial hosts. Hence, a strategy that uses a fusion technique and engineered Escherichia coli strains was employed to improve the expression of soluble rHSA in the bacterial cytoplasm. The solubilities of the b'a' domain of human protein disulphide isomerase (PDIb'a')- and maltose-binding protein (MBP)-tagged rHSA expressed in Origami 2 at 18 °C were notably increased by up to 90.1% and 96%, respectively. A simple and efficient protocol for rHSA purification was established and approximately 9.46 mg rHSA was successfully obtained from a 500-mL culture at 97% purity. However, rHSA was mostly obtained in soluble oligomeric form. By introducing a simple refolding and size-exclusion chromatography step, monomeric rHSA was obtained at 34% yield. Native polyacrylamide gel electrophoresis confirmed the similarity in the molecular weights between E. coli-derived monomeric rHSA and commercial monomeric HSA.