scholarly journals Functional reconstitution of TatB into thylakoidal Tat translocase

2019 ◽  
Author(s):  
Sarah Zinecker ◽  
Mario Jakob ◽  
Ralf Bernd Klösgen
Keyword(s):  
Functional Analysis ◽  
Heterologous Expression ◽  
Protein Transport ◽  
Transmembrane Helix ◽  
Experimental System ◽  
Receptor Complex ◽  
Transport Activity ◽  
E Coli ◽  
Purified Antigen

AbstractWe have established an experimental system for the functional analysis of thylakoidal TatB, a component of the membrane-integral TatBC receptor complex of the thylakoidal Twin-arginine protein transport (Tat1) machinery. For this purpose, the intrinsic TatB activity of isolated pea thylakoids was inhibited by affinity-purified antibodies and substituted by supplementing the assays with TatB protein either obtained by in vitro translation or purified after heterologous expression in E. coli. Tat transport activity of such reconstituted thylakoids, which was analyzed with the authentic Tat substrate pOEC16, reached routinely 20 - 25% of the activity of mock-treated thylakoid vesicles analysed in parallel. In contrast, supplementation of the assays with the purified antigen comprising all but the N-terminal transmembrane helix of thylakoidal TatB did not result in Tat transport reconstitution which confirms that transport relies strictly on the activity of the TatB protein added and is not due to restoration of the intrinsic TatB activity by antibody release. Unexpectedly, even a mutant TatB protein (TatB,E10C) assumed to be incapable of assembling into the TatBC receptor complex showed low but considerable transport reconstitution underlining the sensitivity of the approach and its suitability for further functional mutant analyses. Finally, quantification of TatB demand suggests that TatA and TatB are required in approximately equimolar amounts to achieve Tat-dependent thylakoid transport.

Molecular characterization, heterologous expression and resistance analysis of OsLEA3-1 from Oryza sativa

Biologia ◽  
2014 ◽  
Vol 69 (5) ◽  
Author(s):  
Tingzhang Hu ◽  
Junnian Yang ◽  
Yongwei Yang ◽  
Yingmei Wu
Keyword(s):  
Fusion Protein ◽  
Heterologous Expression ◽  
Abiotic Stresses ◽  
Structure Prediction ◽  
Bioinformatics Analysis ◽  
Secondary Structure Prediction ◽  
Mature Embryo ◽  
Lactate Dehydrogenase Activity ◽  
E Coli

AbstractLate embryogenesis abundant (LEA) proteins in organisms are closely associated with resistance to abiotic stresses. Here we characterized a rice LEA protein, OsLEA3-1, by bioinformatics analysis and heterologous expression in Escherichia coli. Bioinformatics analysis showed that OsLEA3-1 contains a 603-bp open reading frame encoding a putative polypeptide of 200 amino acids, which contains a “LEA_4” motif at positions 5–48 and belongs to a typical group 3 LEA. OsLEA3-1 polypeptide is rich in Ala, Lys, and Thr, but depleted in Cys, Pro, and Trp residues; and is strongly hydrophilic. Secondary structure prediction showed that OsLEA3-1 polypeptide contained an α-helical domain in positions 4-195 but not any β-sheet domain. OsLEA3-1 gene can express in shoot and root of germinating seeds, seedling, panicles, mature embryo, seed, and callus; and was also up-regulated by ultraviolet (UV), heat, cold, salt, and emergency drought. OsLEA3-1 gene was introduced into E. coli. A fusion protein of about 28.03 kDa was expressed in recombinant E. coli cells after the induction by isopropylthio-β-D-galactoside. Compared with control E. coli cells harbouring pET30a, the accumulation of the OsLEA3-1 fusion protein increased the tolerance of the E. coli recombinants under diverse abiotic stresses: high salinity, metal ions, hyperosmotic, heat, and UV radiation. The OsLEA3-1 has the ability to protect the lactate dehydrogenase activity under heating, drying, and MnCl2 treatment in vitro. The findings suggested that the OsLEA3-1 gene may contribute to the ability of adapting to stressful environments of plants.

scholarly journals Characterization and Heterologous Expression of UDP-Glucose 4-Epimerase From a Hericium erinaceus Mutant with High Polysaccharide Production

2021 ◽  
Vol 9 ◽  
Author(s):  
Gen Zou ◽  
Juanbao Ren ◽  
Di Wu ◽  
Henan Zhang ◽  
Ming Gong ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Genetic Manipulation ◽  
Hericium Erinaceus ◽  
E Coli ◽  
Macrophage Activity ◽  
Medicinal Fungus ◽  
Polysaccharide Composition ◽  
High Production ◽  
The Many

Hericium erinaceus is an important medicinal fungus in traditional Chinese medicine because of its polysaccharides and other natural products. Compared terpenoids and polyketides, the analysis of synthetic pathway of polysaccharides is more difficult because of the many genes involved in central metabolism. In previous studies, A6180, encoding a putative UDP-glucose 4-epimerase (UGE) in an H. erinaceus mutant with high production of active polysaccharides, was significantly upregulated. Since there is no reliable genetic manipulation technology for H. erinaceus, we employed Escherichia coli and Saccharomyces cerevisiae to study the function and activity of A6180. The recombinant overexpression vector pET22b-A6180 was constructed for heterologous expression in E. coli. The enzymatic properties of the recombinant protein were investigated. It showed that the recombinant A6180 could strongly convert UDP-α-D-glucose into UDP-α-D-galactose under optimal conditions (pH 6.0, 30°C). In addition, when A6180 was introduced into S. cerevisiae BY4742, xylose was detected in the polysaccharide composition of the yeast transformant. This suggested that the protein coded by A6180 might be a multifunctional enzyme. The generated polysaccharides with a new composition of sugars showed enhanced macrophage activity in vitro. These results indicate that A6180 plays an important role in the structure and activity of polysaccharides. It is a promising strategy for producing polysaccharides with higher activity by introducing A6180 into polysaccharide-producing mushrooms.

scholarly journals Thylakoid-integrated recombinant Hcf106 participates in the chloroplast Twin Arginine Transport (cpTat) system

2018 ◽  
Author(s):  
Qianqian Ma ◽  
Kristen Fite ◽  
Christopher Paul New ◽  
Carole Dabney-Smith
Keyword(s):  
Protein Transport ◽  
Transmembrane Domain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sole Source ◽  
Receptor Complex ◽  
Amphipathic Helix ◽  
Transport Pathway ◽  
Independent Manner ◽  
Arginine Transport

AbstractThe chloroplast Twin arginine transport (cpTat) system distinguishes itself as a protein transport pathway by translocating fully-folded proteins, using the proton-motive force (PMF) as the sole source of energy. The cpTat pathway is evolutionarily conserved with the Tat pathway found in the plasma membrane of many prokaryotes. The cpTat (E. coli) system uses three proteins, Tha4 (TatA), Hcf106 (TatB), and cpTatC (TatC), to form a transient translocase allowing the passage of precursor proteins. Briefly, cpTatC and Hcf106, with Tha4, form the initial receptor complex responsible for precursor protein recognition and binding in an energy-independent manner, while a separate pool of Tha4 assembles with the precursor-bound receptor complex in the presence the PMF. Analysis by blue-native polyacrylamide gel electrophoresis (BN-PAGE) shows that the receptor complex, in the absence of precursor, migrates near 700 kDa and contains cpTatC and Hcf106 with little Tha4 remaining after detergent solubilization. To investigate the role that Hcf106 may play in receptor complex oligomerization and/or stability, systematic cysteine substitutions were made in positions from the N-terminal transmembrane domain to the end of the predicted amphipathic helix of the protein. BN-PAGE analysis allowed us to identify the locations of amino acids in Hcf106 that were critical for interacting with cpTatC. Oxidative cross-linking allowed us to map interactions of the transmembrane domain and amphipathic helix region of Hcf106. In addition, we showed that in vitro expressed, integrated Hcf106 can interact with the precursor signal peptide domain and imported cpTatC, strongly suggesting that a subpopulation of the integrated Hcf106 is participating in competent cpTat complexes.

scholarly journals Widening the bottleneck: Heterologous expression, purification, and characterization of the Ktedonobacter racemifer minimal type II polyketide synthase in Escherichia coli

2020 ◽  
Author(s):  
Joshua G. Klein ◽  
Yang Wu ◽  
Bashkim Kokona ◽  
Louise K. Charkoudian
Keyword(s):  
Heterologous Expression ◽  
Polyketide Synthase ◽  
Analytical Ultracentrifugation ◽  
Acyl Carrier Protein ◽  
Unmet Need ◽  
Type Ii ◽  
E Coli ◽  
Potential Impact

AbstractEnzyme assemblies such as type II polyketide synthases (PKSs) produce a wide array of bioactive secondary metabolites. While the molecules produced by type II PKSs have found remarkable success in the clinic, the biosynthetic prowess of these enzymes has been stymied by: 1) the inability to reconstitute the bioactivity of the minimal PKS enzymes in vitro and 2) limited exploration of type II PKSs from diverse phyla. Towards filling this unmet need, we expressed, purified, and characterized the ketosynthase chain length factor (KSCLF) and acyl carrier protein (ACP) from Ktedonobacter racemifer. Using E. coli as a heterologous host, we obtained soluble proteins in titers representing significant improvements over previous KSCLF heterologous expression efforts. Characterization of these enzymes reveals that KrACP has self-malonylating activity. Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of holo-KrACP and KrKSCLF indicates that these enzymes do not interact in vitro, suggesting that the acylated state of these proteins might play an important role in facilitating biosynthetically relevant interactions. Given the potential impact of obtaining soluble core type II PKS biosynthetic enzymes to enable in vitro characterization studies, these results lay important groundwork for optimizing the interaction between KrKSCLF and KrACP and exploring the biosynthetic potential of other non-actinomycete type II PKSs.

scholarly journals Identification of the amine–polyamine–choline transporter superfamily ‘consensus amphipathic region’ as the target for inactivation of the Escherichia coli GABA transporter GabP by thiol modification reagents. Role of Cys-300 in restoring thiol sensitivity to GabP lacking Cys

1999 ◽  
Vol 339 (3) ◽  
pp. 649-655
Author(s):  
Liaoyuan A. HU ◽  
Steven C. KING
Keyword(s):  
Escherichia Coli ◽  
Transmembrane Helix ◽  
Structural Similarity ◽  
Inhibitory Effect ◽  
Cysteine Residue ◽  
Transport Activity ◽  
Pyridinecarboxylic Acid ◽  
E Coli ◽  
The One

The Escherichia coli γ-aminobutyric acid transporter GabP (gab permease) contains a functionally significant cysteine residue (Cys-300) within its consensus amphipathic region (CAR), a putative channel-forming structure that extends out of transmembrane helix 8 and into the adjoining cytoplasmic loop 8-9 of transporters from the amine-polyamine-choline (APC) superfamily. Here we show that of the five cysteine residues (positions 158, 251, 291, 300 and 443) in the E. coli GabP, Cys-300 is the one that renders the transport activity sensitive to inhibition by thiol modification reagents: whereas substituting Ala for Cys-300 mimics the inhibitory effect of thiol modification, substituting Ala at position 158, 251, 291 or 443 preserves robust transport activity and confers no resistance to thiol inactivation; and whereas the robustly active Cys-300 single-Cys mutant is fully sensitive to thiol modification, other single-Cys mutants (Cys at 158, 251, 291 or 443) exhibit kinetically compromised transport activities that resist further chemical inactivation by thiol reagents. The present study reveals additionally that Cys-300 exhibits (1) sensitivity to hydrophobic thiol reagents, (2) general resistance to bulky (fluorescein 5-maleimide) and/or charged {2-sulphonatoethyl methanethiosulphonate or [2-(trimethylammonium)ethyl] methanethiosulphonate} thiol reagents and (3) a peculiar sensitivity to p-chloromercuribenzenesulphonate (PCMBS). The accessibility of PCMBS to Cys-300 (located midway through the lipid bilayer) might be related to the structural similarity that it shares with guvacine (1,2,3,6-tetrahydro-3-pyridinecarboxylic acid), a transported GabP substrate. These structural requirements for thiol sensitivity provide the first chemical evidence consistent with channel-like access to the polar surface of the CAR, a physical configuration that might provide a basis for understanding how this region impacts the function of APC transporters generally [Closs, Lyons, Kelly and Cunningham (1993) J. Biol. Chem. 268, 20796-20800] and the gab permease particularly [Hu and King (1998) Biochem. J. 300, 771-776].

scholarly journals Combining ancestral reconstruction with folding-landscape simulations to engineer heterologous protein expression

2021 ◽  
Author(s):  
Gloria Gamiz-Arco ◽  
Valeria A. Risso ◽  
Eric A. Gaucher ◽  
Jose A. Gavira ◽  
Athi N. Naganathan ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
3D Structure ◽  
Sequence Divergence ◽  
Redox Activity ◽  
Structure Stability ◽  
Heterologous Protein Expression ◽  
Ancestral Reconstruction ◽  
E Coli ◽  
Statistical Mechanical Model

Obligate symbionts exhibit high evolutionary rates and extensive sequence divergence. Here, we use the thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiont of flashlight fish, to explore evolutionary and engineering aspects of protein folding in heterologous hosts. The symbiont protein is a standard thioredoxin in terms of 3D-structure, stability and redox activity. However, its refolding in vitro is very slow and its expression in E. coli leads to insoluble protein. By contrast, resurrected Precambrian thioredoxins express efficiently in E. coli, plausibly reflecting an ancient adaptation to unassisted folding. We have used a statistical-mechanical model of the folding landscape to guide back-to-ancestor engineering of the symbiont protein. Remarkably, we find that the efficiency of heterologous expression correlates with the in vitro refolding rate and that the ancestral expression efficiency can be achieved with only 1-2 back-to-ancestor replacements. These results demonstrate a sequence-engineering approach to rescue inefficient heterologous expression, a major biotechnological bottleneck.

scholarly journals 18-Hydroxydolabella-3,7-diene synthase – a diterpene synthase from Chitinophaga pinensis

2017 ◽  
Vol 13 ◽  
pp. 1770-1780 ◽  
Author(s):  
Jeroen S Dickschat ◽  
Jan Rinkel ◽  
Patrick Rabe ◽  
Arman Beyraghdar Kashkooli ◽  
Harro J Bouwmeester
Keyword(s):  
Heterologous Expression ◽  
Nicotiana Benthamiana ◽  
Terpene Synthase ◽  
In Planta ◽  
Isotopic Labelling ◽  
E Coli ◽  
Diterpene Synthase ◽  
Diterpene Alcohol ◽  
Isotopic Labelling Experiments

The product obtained in vitro from a diterpene synthase encoded in the genome of the bacterium Chitinophaga pinensis, an enzyme previously reported to have germacrene A synthase activity during heterologous expression in Escherichia coli, was identified by extensive NMR-spectroscopic methods as 18-hydroxydolabella-3,7-diene. The absolute configuration of this diterpene alcohol and the stereochemical course of the terpene synthase reaction were addressed by isotopic labelling experiments. Heterologous expression of the diterpene synthase in Nicotiana benthamiana resulted in the production of 18-hydroxydolabella-3,7-diene also in planta, while the results from the heterologous expression in E. coli were shown to be reproducible, revealing that the expression of one and the same terpene synthase in different heterologous hosts may yield different terpene products.

EVALUATION OF TISSUE STEROID BINDING IN VITRO

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S223-S246 ◽  
Author(s):  
C. R. Wira ◽  
H. Rochefort ◽  
E. E. Baulieu
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Experimental System ◽  
Specific Protein ◽  
Coupling Mechanism ◽  
Target Tissue ◽  
Protein Binding Sites ◽  
Definition Of ◽  
Hormone Specificity

ABSTRACT The definition of a RECEPTOR* in terms of a receptive site, an executive site and a coupling mechanism, is followed by a general consideration of four binding criteria, which include hormone specificity, tissue specificity, high affinity and saturation, essential for distinguishing between specific and nonspecific binding. Experimental approaches are proposed for choosing an experimental system (either organized or soluble) and detecting the presence of protein binding sites. Techniques are then presented for evaluating the specific protein binding sites (receptors) in terms of the four criteria. This is followed by a brief consideration of how receptors may be located in cells and characterized when extracted. Finally various examples of oestrogen, androgen, progestagen, glucocorticoid and mineralocorticoid binding to their respective target tissues are presented, to illustrate how researchers have identified specific corticoid and mineralocorticoid binding in their respective target tissue receptors.

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