ABSTRACT
INTRODUCTION: In the biopharmaceutical industry, Escherichia coli is one of the preferred expression hosts for large-scale production of therapeutic proteins. Although increasing the product yield is important, product quality is a major factor in this industry because greatest productivity does not always correspond with the highest quality of the produced protein. While some post-translational modifications, such as disulphide bonds, are required to achieve the biologically active conformation, others may have a negative impact on the product's activity, effectiveness, and/or safety. Therefore, they are classified as product associated impurities, and they represent a crucial quality parameter for regulatory authorities. RESULTS: In this study, fermentation conditions of two widely employed industrial E. coli strains, BL21 and W3110 are compared for recombinant protein production of a single-chain variable fragment (scFv) in an industrial setting. We found that the BL21 strain produces more soluble scFv than the W3110 strain, even though W3110 produces more recombinant protein in total. A quality assessment on the scFv recovered from the supernatant was then performed. Unexpectedly, even when our scFv is correctly disulphide bonded and cleaved from its signal peptide in both strains, the protein shows charge heterogeneity with up to seven distinguishable variants on cation exchange chromatography. Biophysical characterization confirmed the presence of altered conformations of the two main charged variants. CONCLUSIONS: The findings indicated that BL21 is more productive for this specific scFv than W3110. When assessing product quality, a distinctive profile of the protein was found which was independent of the E. coli strain. This suggests that alterations are present in the recovered product although the exact nature of them could not be determined. This similarity between the two strains' generated products also serves as a sign of their interchangeability. This study encourages the development of innovative, fast, and inexpensive techniques for the detection of heterogeneity while also provoking a debate about whether intact mass spectrometry-based analysis of the protein of interest is sufficient to detect heterogeneity in a product.
Subject(s)
Escherichia coli Proteins , Single-Chain Antibodies , Escherichia coli/metabolism , Single-Chain Antibodies/genetics , Single-Chain Antibodies/metabolism , Recombinant Proteins , Escherichia coli Proteins/metabolism , Disulfides/metabolismABSTRACT
Most Escherichia coli overexpression vectors used for recombinant protein production (RPP) depend on organic inducers, for example, sugars or simple conjugates. However, these can be expensive and, sometimes, chemically unstable. To simplify this and to cut the cost of RPP, we have developed vectors controlled by the Escherichia coli nitrate-responsive NarL transcription activator protein, which use nitrate, a cheap, stable, and abundant inorganic ion, to induce high-level controlled RPP. We show that target proteins, such as green fluorescent protein, human growth hormone, and single-chain variable region antibody fragments can be expressed to high levels using our promoter systems. As nitrate levels are high in many commercial fertilizers, we demonstrate that controlled RPP can be achieved using readily available and inexpensive garden products.
Subject(s)
Escherichia coli Proteins , Base Sequence , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Humans , Nitrates/metabolism , Operon , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has led to a pandemic, that continues to be a huge public health burden. Despite the availability of vaccines, there is still a need for small-molecule antiviral drugs. In an effort to identify novel and drug-like hit matter that can be used for subsequent hit-to-lead optimization campaigns, we conducted a high-throughput screening of a 160 K compound library against SARS-CoV-2, yielding a 1-heteroaryl-2-alkoxyphenyl analog as a promising hit. Antiviral profiling revealed this compound was active against various beta-coronaviruses and preliminary mode-of-action experiments demonstrated that it interfered with viral entry. A systematic structure-activity relationship (SAR) study demonstrated that a 3- or 4-pyridyl moiety on the oxadiazole moiety is optimal, whereas the oxadiazole can be replaced by various other heteroaromatic cycles. In addition, the alkoxy group tolerates some structural diversity.
Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Heterocyclic Compounds/pharmacology , SARS-CoV-2/drug effects , Virus Replication/drug effects , Animals , Chlorocebus aethiops , High-Throughput Screening Assays , Microbial Sensitivity Tests , Structure-Activity Relationship , Vero CellsABSTRACT
Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin-arginine translocase (Tat). The Tat machinery exports folded and cofactor-containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality-control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Escherichia coli Tat system to recognize and translocate de novo-designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one, or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b-induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system's quality-control mechanism.
Subject(s)
Bacterial Proteins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Hemeproteins/metabolism , Membrane Transport Proteins/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Circular Dichroism , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Heme-Binding Proteins , Hemeproteins/chemistry , Membrane Transport Proteins/chemistry , Methylamines/metabolism , Models, Molecular , Oxidoreductases, N-Demethylating/metabolism , Periplasm/metabolism , Protein Folding , Protein Sorting Signals , Protein Stability , Protein Transport , Proton Magnetic Resonance Spectroscopy , Substrate Specificity , TemperatureABSTRACT
The Escherichia coli lac operon promoter is widely used as a tool to control recombinant protein production in bacteria. Here, we give a brief review of how it functions, how it is regulated, and how, based on this knowledge, a suite of lac promoter derivatives has been developed to give a controlled expression that is suitable for diverse biotechnology applications.
Subject(s)
Escherichia coli/metabolism , Lac Operon/genetics , Promoter Regions, Genetic/genetics , Escherichia coli/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transcription, Genetic/geneticsABSTRACT
The large-scale production and isolation of recombinant protein is a central element of the biotechnology industry and many of the products have proved extremely beneficial for therapeutic medicine. Escherichia coli is the microorganism of choice for the expression of heterologous proteins for therapeutic application, and a range of high-value proteins have been targeted to the periplasm using the well characterized Sec protein export pathway. More recently, the ability of the second mainstream protein export system, the twin-arginine translocase, to transport fully-folded proteins into the periplasm of not only E. coli, but also other Gram-negative bacteria, has captured the interest of the biotechnology industry. In this study, we have used a novel approach to block the export of a heterologous Tat substrate in the later stages of the export process, and thereby generate a single-span membrane protein with the soluble domain positioned on the periplasmic side of the inner membrane. Biochemical and immuno-electron microscopy approaches were used to investigate the export of human growth hormone by the twin-arginine translocase, and the generation of a single-span membrane-embedded variant. This is the first time that a bonafide biotechnologically relevant protein has been exported by this machinery and visualized directly in this manner. The data presented here demonstrate a novel method for the production of single-span membrane proteins in E. coli.
Subject(s)
Escherichia coli/metabolism , Human Growth Hormone/metabolism , Industrial Microbiology , Escherichia coli/cytology , Human Growth Hormone/analysis , Humans , Industrial Microbiology/methods , Protein Sorting Signals , Protein Transport , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/metabolism , Solubility , Twin-Arginine-Translocation System/analysis , Twin-Arginine-Translocation System/metabolismABSTRACT
Escherichia coli is a heavily used platform for the production of biotherapeutic and other high-value proteins, and a favored strategy is to export the protein of interest to the periplasm to simplify downstream processing and facilitate disulfide bond formation. The Sec pathway is the standard means of transporting the target protein but it is unable to transport complex or rapidly folding proteins because the Sec system can only transport proteins in an unfolded state. The Tat system also operates to transport proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Here, we have tested the Tat system's full potential for the production of biotherapeutics for the first time using fed-batch fermentation. We expressed human growth hormone (hGH) with a Tat signal peptide in E. coli W3110 "TatExpress" strains that contain elevated levels of the Tat apparatus. This construct contained four amino acids from TorA at the hGH N-terminus as well as the initiation methionine from hGH, which is removed in vivo. We show that the protein is efficiently exported to the periplasm during extended fed-batch fermentation, to the extent that it is by far the most abundant protein in the periplasm. The protein was shown to be homogeneous, disulfide bonded, and active. The bioassay showed that the yields of purified periplasmic hGH are 5.4 g/L culture whereas an enzyme-linked immunosorbent assay gave a figure of 2.39 g/L. Separate analysis of a TorA signal peptide linked to hGH construct lacking any additional amino acids likewise showed efficient export to the periplasm, although yields were approximately two-fold lower.
Subject(s)
Escherichia coli/metabolism , Human Growth Hormone/biosynthesis , Periplasm/metabolism , Protein Folding , Protein Sorting Signals , Recombinant Fusion Proteins/biosynthesis , Escherichia coli/genetics , Human Growth Hormone/genetics , Humans , Periplasm/genetics , Recombinant Fusion Proteins/geneticsABSTRACT
BACKGROUND: The Twin-arginine translocation (Tat) pathway of Escherichia coli has great potential for the export of biopharmaceuticals to the periplasm due to its ability to transport folded proteins, and its proofreading mechanism that allows correctly folded proteins to translocate. Coupling the Tat-dependent protein secretion with the formation of disulfide bonds in the cytoplasm of E. coli CyDisCo provides a powerful platform for the production of industrially challenging proteins. In this study, we investigated the effects on the E. coli cells of exporting a folded substrate (scFv) to the periplasm using a Tat signal peptide, and the effects of expressing an export-incompetent misfolded variant. RESULTS: Cell growth is decreased when either the correctly folded or misfolded scFv is expressed with a Tat signal peptide. However, only the production of misfolded scFv leads to cell aggregation and formation of inclusion bodies. The comprehensive proteomic analysis revealed that both conditions, recombinant protein overexpression and misfolded protein accumulation, lead to downregulation of membrane transporters responsible for protein folding and insertion into the membrane while upregulating the production of chaperones and proteases involved in removing aggregates. These conditions also differentially affect the production of transcription factors and proteins involved in DNA replication. The most distinct stress response observed was the cell aggregation caused by elevated levels of antigen 43. Finally, Tat-dependent secretion causes an increase in tatA expression only after induction of protein expression, while the subsequent post-induction analysis revealed lower tatA and tatB expression levels, which correlate with lowered TatA and TatB protein abundance. CONCLUSIONS: The study identified characteristic changes occurring as a result of the production of both a folded and a misfolded protein, but also highlights an exclusive unfolded stress response. Countering and compensating for these changes may result in higher yields of pharmaceutically relevant proteins exported to the periplasm.
Subject(s)
Escherichia coli/genetics , Oxidative Stress , Protein Folding , Proteome , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Membrane Transport Proteins , Protein Sorting Signals , Protein Transport , Proteomics , Recombinant Fusion Proteins/chemistry , Single-Chain Antibodies/chemistryABSTRACT
Disruption of interleukin-13 (IL-13) signaling with large molecule antibody therapies has shown promise in diseases of allergic inflammation. Given that IL-13 recruits several members of the Janus Kinase family (JAK1, JAK2, and TYK2) to its receptor complex, JAK inhibition may offer an alternate small molecule approach to disrupting IL-13 signaling. Herein we demonstrate that JAK1 is likely the isoform most important to IL-13 signaling. Structure-based design was then used to improve the JAK1 potency of a series of previously reported JAK2 inhibitors. The ability to impede IL-13 signaling was thereby significantly improved, with the best compounds exhibiting single digit nM IC50's in cell-based assays dependent upon IL-13 signaling. Appropriate substitution was further found to influence inhibition of a key off-target, LRRK2. Finally, the most potent compounds were found to be metabolically labile, which makes them ideal scaffolds for further development as topical agents for IL-13 mediated diseases of the lungs and skin (for example asthma and atopic dermatitis, respectively).
Subject(s)
Dermatitis, Atopic/genetics , Interleukin-13/metabolism , Janus Kinase 1/antagonists & inhibitors , Janus Kinase 2/antagonists & inhibitors , Humans , Signal TransductionABSTRACT
The Tat system preferentially transports correctly folded proteins across the bacterial membrane although little is known of the proofreading mechanism. Most research has focused on TatABC systems from Gram-negative bacteria, especially Escherichia coli, and much less is known of the TatAC-type systems from Gram-positive organisms. We have previously shown that the Bacillus subtilis TatAdCd system is functional in an E. coli tat null background and able to transport TorA-GFP and native TorA (TMAO reductase); here, we examined its ability to transport other proteins bearing a TorA signal sequence. We show that whereas E. coli TatABC transports a wide range of biotherapeutics including human growth hormone, interferon α2b, a VH domain protein and 2 different scFvs, TatAdCd transports the scFvs but completely rejects the other proteins. The system also rejects two native E. coli substrates, NrfC and FhuD. Moreover, we have shown that TatABC will transport a wide range of folded scFv variants with the surface altered to incorporate multiple salt bridges, charged residues (5 glutamate, lysine or arginine), or hydrophobic residues (up to 6 leucines). In contrast, TatAdCd completely rejects many of these variants including those with 5 or 6 added Leu residues. The combined data show that the TatABC and TatAdCd systems have very different substrate selectivities, with the TatAdCd system displaying an extreme level of selectivity when compared to the E. coli system. The data also provide a preliminary suggestion that TatAdCd may not tolerate substrates that contain surface domains with a level of hydrophobicity above a certain threshold.
Subject(s)
Bacillus subtilis/metabolism , Escherichia coli Proteins/metabolism , Membrane Transport Proteins/metabolism , Oxidoreductases, N-Demethylating/metabolism , Single-Chain Antibodies/metabolism , Amino Acid Sequence , Bacillus subtilis/genetics , Biological Transport , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Gene Expression , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Human Growth Hormone/chemistry , Human Growth Hormone/metabolism , Hydrophobic and Hydrophilic Interactions , Interferon alpha-2 , Interferon-alpha/chemistry , Interferon-alpha/metabolism , Membrane Transport Proteins/genetics , Oxidoreductases, N-Demethylating/genetics , Protein Folding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Single-Chain Antibodies/chemistry , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/metabolism , Static Electricity , Substrate SpecificityABSTRACT
The oral S1PR1 agonist ponesimod demonstrated substantial efficacy in a phase II clinical trial of psoriasis. Unfortunately, systemic side effects were observed, which included lymphopenia and transient bradycardia. We sought to develop a topical soft-drug S1PR1 agonist with an improved therapeutic index. By modifying ponesimod, we discovered an ester series of S1PR agonists. To increase metabolic instability in plasma we synthesised esters described as specific substrates for paraoxonase and butyrylcholinesterases, esterases present in human plasma.
Subject(s)
Drug Discovery , Receptors, Lysosphingolipid/drug effects , Thiazoles/pharmacology , Administration, Topical , Aryldialkylphosphatase/blood , Chromatography, High Pressure Liquid , Chromatography, Reverse-Phase , Esterases/blood , Esterases/metabolism , Humans , Skin/enzymology , Solubility , Sphingosine-1-Phosphate Receptors , Structure-Activity Relationship , Thiazoles/administration & dosageABSTRACT
The twin-arginine translocation (Tat) system is an integral membrane protein complex that accomplishes the remarkable feat of transporting large, fully folded polypeptides across the inner membrane of bacteria, into the periplasm. In Escherichia coli, Tat comprises three membrane proteins: TatA, TatB and TatC. How these proteins arrange themselves in the inner membrane to permit passage of Tat substrates, whilst maintaining membrane integrity, is still poorly understood. TatA is the most abundant component of this complex and facilitates assembly of the transport mechanism. We have utilised immunogold labelling in combination with array tomography to gain insight into the localisation and distribution of the TatA protein in E. coli cells. We show that TatA exhibits a uniform distribution throughout the inner membrane of E. coli and that altering the expression of TatBC shows a previously uncharacterised distribution of TatA in the inner membrane. Array tomography was used to provide our first insight into this altered distribution of TatA in three-dimensional space, revealing that this protein forms linear clusters in the inner membrane of E. coli upon increased expression of TatBC. This is the first indication that TatA organisation in the inner membrane alters in response to changes in Tat subunit stoichiometry.
Subject(s)
Escherichia coli Proteins/ultrastructure , Imaging, Three-Dimensional/methods , Membrane Transport Proteins/ultrastructure , Microscopy, Immunoelectron/methods , Multiprotein Complexes/ultrastructure , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli/ultrastructure , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Operon , Protein Binding , Protein TransportABSTRACT
Cyanobacteria exhibit a complex form of membrane differentiation that sets them apart from most bacteria. Many processes take place in the plasma membrane, but photosynthetic light capture, electron transport and ATP synthesis take place in an abundant internal thylakoid membrane. This review considers how this system of subcellular compartmentalisation is maintained, and how proteins are directed towards the various subcompartments--specifically the plasma membrane, periplasm, thylakoid membrane and thylakoid lumen. The involvement of Sec-, Tat- and signal recognition particle- (SRP)-dependent protein targeting pathways is discussed, together with the possible involvement of a so-called 'spontaneous' pathway for the insertion of membrane proteins, previously characterised for chloroplast thylakoid membrane proteins. An intriguing aspect of cyanobacterial cell biology is that most contain only a single set of genes encoding Sec, Tat and SRP components, yet the proteomes of the plasma and thylakoid membranes are very different. The implications for protein sorting mechanisms are considered. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Prof Conrad Mullineaux.
Subject(s)
Bacterial Proteins/metabolism , Cyanobacteria/metabolism , Thylakoids/metabolism , Bacterial Proteins/genetics , Cyanobacteria/genetics , Protein Transport/physiology , Thylakoids/geneticsABSTRACT
The Tat system transports folded proteins across the bacterial plasma membrane, and in Escherichia coli preferentially transports correctly-folded proteins. Little is known of the mechanism by which Tat proofreads a substrate's conformational state, and in this study we have addressed this question using a heterologous single-chain variable fragment (scFv) with a defined structure. We introduced mutations to surface residues while leaving the folded structure intact, and also tested the importance of conformational flexibility. We show that while the scFv is stably folded and active in the reduced form, formation of the 2 intra-domain disulphide bonds enhances Tat-dependent export 10-fold, indicating Tat senses the conformational flexibility and preferentially exports the more rigid structure. We further show that a 26-residue unstructured tail at the C-terminus blocks export, suggesting that even this short sequence can be sensed by the proofreading system. In contrast, the Tat system can tolerate significant changes in charge or hydrophobicity on the scFv surface; substitution of uncharged residues by up to 3 Lys-Glu pairs has little effect, as has the introduction of up to 5 Lys or Glu residues in a confined domain, or the introduction of a patch of 4 to 6 Leu residues in a hydrophilic region. We propose that the proofreading system has evolved to sense conformational flexibility and detect even very transiently-exposed internal regions, or the presence of unfolded peptide sections. In contrast, it tolerates major changes in surface charge or hydrophobicity.
Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Membrane Transport Proteins/chemistry , Single-Chain Antibodies/chemistry , Transgenes , Amino Acid Motifs , Binding Sites , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Models, Molecular , Mutation , Plasmids/chemistry , Plasmids/metabolism , Protein Binding , Protein Folding , Protein Interaction Domains and Motifs , Protein Sorting Signals , Protein Structure, Secondary , Protein Transport , Single-Chain Antibodies/genetics , Single-Chain Antibodies/metabolism , Static Electricity , Substrate SpecificityABSTRACT
Photosynthesis drives the production of ATP and NADPH, and acts as a source of carbon for primary metabolism. NADPH is also used in the production of many natural bioactive compounds. These are usually synthesized in low quantities and are often difficult to produce by chemical synthesis due to their complex structures. Some of the crucial enzymes catalyzing their biosynthesis are the cytochromes P450 (P450s) situated in the endoplasmic reticulum (ER), powered by electron transfers from NADPH. Dhurrin is a cyanogenic glucoside and its biosynthesis involves a dynamic metabolon formed by two P450s, a UDP-glucosyltransferase (UGT) and a P450 oxidoreductase (POR). Its biosynthetic pathway has been relocated to the chloroplast where ferredoxin, reduced through the photosynthetic electron transport chain, serves as an efficient electron donor to the P450s, bypassing the involvement of POR. Nevertheless, translocation of the pathway from the ER to the chloroplast creates other difficulties, such as the loss of metabolon formation and intermediate diversion into other metabolic pathways. We show here that co-localization of these enzymes in the thylakoid membrane leads to a significant increase in product formation, with a concomitant decrease in off-pathway intermediates. This was achieved by exchanging the membrane anchors of the dhurrin pathway enzymes to components of the Twin-arginine translocation pathway, TatB and TatC, which have self-assembly properties. Consequently, we show 5-fold increased titers of dhurrin and a decrease in the amounts of intermediates and side products in Nicotiana benthamiana. Further, results suggest that targeting the UGT to the membrane is a key factor to achieve efficient substrate channeling.
Subject(s)
Chloroplast Proteins , Cytochrome P-450 Enzyme System , Membrane Proteins , Nicotiana , Nitriles/metabolism , Plant Proteins , Plants, Genetically Modified , Recombinant Fusion Proteins , Thylakoids , Chloroplast Proteins/biosynthesis , Chloroplast Proteins/genetics , Cytochrome P-450 Enzyme System/biosynthesis , Cytochrome P-450 Enzyme System/genetics , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Plant Proteins/biosynthesis , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Thylakoids/genetics , Thylakoids/metabolism , Nicotiana/genetics , Nicotiana/metabolismABSTRACT
Numerous high-value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec-based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super-secreting strains by introducing the strong inducible bacterial promoter, ptac, upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g., W3110 and BL21). This modification significantly improves the Tat-dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1 hr induction. TatExpress strains accumulate in excess of 30 mg L-1 periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains.
Subject(s)
Escherichia coli/genetics , Escherichia coli/metabolism , Gene Products, tat/genetics , Genetic Enhancement/methods , Growth Hormone/biosynthesis , Periplasm/metabolism , Secretory Pathway/genetics , Growth Hormone/genetics , Growth Hormone/isolation & purification , Humans , Metabolic Networks and Pathways/genetics , Promoter Regions, Genetic/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
Numerous therapeutic proteins are expressed in Escherichia coli and targeted to the periplasm in order to facilitate purification and enable disulfide bond formation. Export is normally achieved by the Sec pathway, which transports proteins through the plasma membrane in a reduced, unfolded state. The Tat pathway is a promising alternative means of export, because it preferentially exports correctly folded proteins; however, the reducing cytoplasm of standard strains has been predicted to preclude export by Tat of proteins that contain disulfide bonds in the native state because, in the reduced state, they are sensed as misfolded and rejected. Here, we have tested a series of disulfide-bond containing biopharmaceuticals for export by the Tat pathway in CyDisCo strains that do enable disulfide bond formation in the cytoplasm. We show that interferon α2b, human growth hormone (hGH) and two antibody fragments are exported with high efficiency; surprisingly, however, they are efficiently exported even in the absence of cytoplasmic disulfide formation. The exported proteins acquire disulfide bonds in the periplasm, indicating that the normal disulfide oxidation machinery is able to act on the proteins. Tat-dependent export of hGH proceeds even when the disulfide bonds are removed by substitution of the Cys residues involved, suggesting that these substrates adopt tertiary structures that are accepted as fully-folded by the Tat machinery.
Subject(s)
Disulfides/metabolism , Escherichia coli Proteins/physiology , Human Growth Hormone/metabolism , Immunoglobulin Fragments/metabolism , Interferon-alpha/metabolism , Membrane Transport Proteins/physiology , Periplasm/metabolism , Amino Acid Sequence , Antibodies/chemistry , Antibodies/metabolism , Disulfides/chemistry , Escherichia coli/metabolism , Humans , Interferon alpha-2 , Metabolic Networks and Pathways , Molecular Sequence Data , Organisms, Genetically Modified , Oxidation-Reduction , Protein Transport , Recombinant Proteins/metabolismABSTRACT
Microalgae have emerged as potentially powerful platforms for the production of recombinant proteins and high-value products. Chlamydomonas reinhardtii is a potentially important host species due to the range of genetic tools that have been developed for this unicellular green alga. Transformation of the chloroplast genome offers important advantages over nuclear transformation, and a wide range of recombinant proteins have now been expressed in the chloroplasts of C. reinhardtii strains. This is often done in cell wall-deficient mutants that are easier to transform. However, only a single study has reported growth data for C. reinhardtii grown at pilot scale, and the growth of cell wall-deficient strains has not been reported at all. Here, we report the first pilot-scale growth study for transgenic, cell wall-deficient C. reinhardtii strains. Strains expressing a cytochrome P450 (CYP79A1) or bifunctional diterpene synthase (cis-abienol synthase, TPS4) were grown for 7 days under mixotrophic conditions in a Tris-acetate-phosphate medium. The strains reached dry cell weights of 0.3 g/L within 3-4 days with stable expression levels of the recombinant proteins during the whole upscaling process. The strains proved to be generally robust, despite the cell wall-deficient phenotype, but grew poorly under phototrophic conditions. The data indicate that cell wall-deficient strains may be highly amenable for transformation and suitable for commercial-scale operations under mixotrophic growth regimes.
Subject(s)
Chlamydomonas reinhardtii/genetics , Chloroplasts/genetics , Cytochrome P-450 Enzyme System/genetics , Glucosyltransferases/genetics , Recombinant Proteins/genetics , Cell Wall/genetics , Cell Wall/metabolism , Chlamydomonas reinhardtii/growth & development , Cytochrome P-450 Enzyme System/metabolism , Gene Transfer Techniques , Glucosyltransferases/metabolism , Recombinant Proteins/biosynthesis , Transformation, Genetic/geneticsABSTRACT
The twin-arginine translocation (Tat) system accomplishes the remarkable feat of translocating large - even dimeric - proteins across tightly sealed energy-transducing membranes. All of the available evidence indicates that it is unique in terms of both structure and mechanism; however its very nature has hindered efforts to probe the core translocation events. At the heart of the problem is the fact that two large sub-complexes are believed to coalesce to form the active translocon, and 'capturing' this translocation event has been too difficult. Nevertheless, studies on the individual components have come a long way in recent years, and structural studies have reached the point where educated guesses can be made concerning the most interesting aspects of Tat. In this article we review these studies and the emerging ideas in this field. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
Subject(s)
Escherichia coli Proteins/genetics , Membrane Transport Proteins/genetics , Protein Sorting Signals/genetics , Protein Transport/genetics , Signal Transduction/genetics , Arginine/chemistry , Arginine/metabolism , Bacterial Translocation , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Protein FoldingABSTRACT
The Tat system transports folded proteins across the bacterial plasma membrane. The mechanism is believed to involve coalescence of a TatC-containing unit with a separate TatA complex, but the full translocation complex has never been visualised and the assembly process is poorly defined. We report the analysis of the Bacillus subtilis TatAyCy system, which occurs as separate TatAyCy and TatAy complexes at steady state, using single-particle electron microscopy (EM) and advanced atomic force microscopy (AFM) approaches. We show that a P2A mutation in the TatAy subunit leads to apparent super-assembly of Tat complexes. Purification of TatCy-containing complexes leads to a large increase in the TatA:TatC ratio, suggesting that TatAy(P2A) complexes may have attached to the TatAyCy complex. EM and AFM analyses show that the wild-type TatAyCy complex purifies as roughly spherical complexes of 9-16nm diameter, whereas the P2A mutation leads to accumulation of large (up to 500nm long) fibrils that are chains of numerous complexes. Time lapsed AFM imaging, recorded on fibrils under liquid, shows that they adopt a variety of tightly curved conformations, with radii of curvature of 10-12nm comparable to the size of single TatAy(P2A) complexes. The combined data indicate that the mutation leads to super-assembly of TatAy(P2A) complexes and we propose that an individual TatAy(P2A) complex assembles initially with a TatAy(P2A)Cy complex, after which further TatAy(P2A) complexes attach to each other. The data further suggest that the N-terminal extracytoplasmic domain of TatAy plays an essential role in Tat complex interactions.