scholarly journals Characterization of Soluble, Disulfide Bond-Stabilized, Prokaryotically Expressed Human Thyrotropin Receptor Ectodomain*

Endocrinology ◽  
1997 ◽  
Vol 138 (2) ◽  
pp. 588-593 ◽  
Author(s):  
Y. Bobovnikova ◽  
P. N. Graves ◽  
H. Vlase ◽  
T. F. Davies
Keyword(s):  
Amino Acids ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Thioredoxin Reductase ◽  
Biologically Active ◽  
Receptor Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fusion Partner ◽  
E Coli ◽  
Recombinant Receptor

Abstract To study the interaction of TSH receptor (TSHR) autoantibodies with receptor protein, it is necessary first to express the receptor in the proper conformation including the formation of correct disulfide bridges. However, the reducing environment of the Escherichia coli (E. coli) cytoplasm prevents the generation of protein disulfide bonds and limits the solubility and immunoreactivity of recombinant human TSHR (hTSHR) products. To circumvent these limitations, hTSHR complementary DNA encoding the extracellular domain (hTSHR-ecd; amino acids 21–415) was inserted into the vector pGEX-2TK by directional cloning and used to transform the thioredoxin reductase mutant strain of E. coli (Ad494), which allowed formation of disulfide bonds in the cytoplasm. After induction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTSHR amino acids 21–35. This showed that over 50% of the expressed hTSHR-ecd was soluble in contrast to expression in a wild-type E. coli (strain αF′), where the majority of the recombinant receptor was insoluble. The soluble recombinant receptor was affinity purified and characterized. Under nonreducing SDS-PAGE conditions, the soluble hTSHR-ecd migrated as refolded, disulfide bond-stabilized, multimeric species, whose formation was independent of fusion partner protein. This product was found to be biologically active as evidenced by the inhibition of the binding of 125I-TSH to the full-length hTSHR expressed in transfected CHO cells and was used to develop a competitive capture enzyme-linked immunosorbent assay for mapping of hTSHR antibody epitopes. Hence, hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation was found to be highly soluble and biologically relevant.

scholarly journals Reconstitution in Proteoliposomes of the Recombinant Human Riboflavin Transporter 2 (SLC52A2) Overexpressed in E. coli

2019 ◽  
Vol 20 (18) ◽  
pp. 4416 ◽  
Author(s):  
Lara Console ◽  
Maria Tolomeo ◽  
Matilde Colella ◽  
Maria Barile ◽  
Cesare Indiveri
Keyword(s):  
Cardiovascular Disease ◽  
Amino Acids ◽  
Risk Factor ◽  
Biologically Active ◽  
Suitable Model ◽  
Native Protein ◽  
E Coli ◽  
Few Data ◽  
Riboflavin Transport

Background: the SLC52A2 gene encodes for the riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed. It mediates the transport of Riboflavin across cell membranes. Riboflavin plays a crucial role in cells since its biologically active forms, FMN and FAD, are essential for the metabolism of carbohydrates, amino acids, and lipids. Mutation of the Riboflavin transporters is a risk factor for anemia, cancer, cardiovascular disease, neurodegeneration. Inborn mutations of SLC52A2 are associated with Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset. In spite of the important metabolic and physio/pathological role of this transporter few data are available on its function and regulation. Methods: the human recombinant RFVT2 has been overexpressed in E. coli, purified and reconstituted into proteoliposomes in order to characterize its activity following the [3H]Riboflavin transport. Results: the recombinant hRFVT2 showed a Km of 0.26 ± 0.07 µM and was inhibited by lumiflavin, FMN and Mg2+. The Riboflavin uptake was also regulated by Ca2+. The native protein extracted from fibroblast and reconstituted in proteoliposomes also showed inhibition by FMN and lumiflavin. Conclusions: proteoliposomes represent a suitable model to assay the RFVT2 function. It will be useful for screening the mutation of RFVT2.

scholarly journals Optimised production of disulfide-bonded fungal effectors in E. coli using CyDisCo and FunCyDisCo co-expression approaches

2021 ◽  
Author(s):  
Daniel Yu ◽  
Megan A Outram ◽  
Emma Creen ◽  
Ashley Smith ◽  
Yi-Chang Sung ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Fungal Pathogens ◽  
Sequence Similarity ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Efficient Production ◽  
E Coli ◽  
Functional Studies ◽  
Folded Structure

Effectors are a key part of the arsenal of plant pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognised by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterise effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a co-expression system within the heterologous host E. coli termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalised co-expression approach termed FunCyDisCo (Fungi-CyDisCo) can significantly improve the production yields of numerous disulfide bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallisation of several of these reported fungal effectors. We suggest this approach could be broadly useful in the investigation of the function and recognition of a broad range of disulfide-bond containing effectors.

scholarly journals Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1157-1163 ◽  
Author(s):  
EA Barron-Casella ◽  
TS Kickler ◽  
OC Rogers ◽  
JF Casella
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Disulfide Bonds ◽  
Affinity Purification ◽  
Platelet Glycoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Glutathione S Transferase ◽  
Amino Terminal ◽  
Posttransfusion Purpura

Abstract The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

scholarly journals Enhancing protein stability with extended disulfide bonds

2016 ◽  
Vol 113 (21) ◽  
pp. 5910-5915 ◽  
Author(s):  
Tao Liu ◽  
Yan Wang ◽  
Xiaozhou Luo ◽  
Jack Li ◽  
Sean A. Reed ◽  
...  
Keyword(s):  
Amino Acids ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Building Blocks ◽  
Side Chain ◽  
Cross Linking ◽  
Noncanonical Amino Acids ◽  
Genetic Encoding ◽  
Selection Experiments ◽  
Significant Distance

Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a library of random β-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9 °C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.

scholarly journals Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1157-1163 ◽  
Author(s):  
EA Barron-Casella ◽  
TS Kickler ◽  
OC Rogers ◽  
JF Casella
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Disulfide Bonds ◽  
Affinity Purification ◽  
Platelet Glycoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Glutathione S Transferase ◽  
Amino Terminal ◽  
Posttransfusion Purpura

The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

scholarly journals Functional Heterologous Expression of Mature Lipase LipA from Pseudomonas aeruginosa PSA01 in Escherichia coli SHuffle and BL21 (DE3): Effect of the Expression Host on Thermal Stability and Solvent Tolerance of the Enzyme Produced

2020 ◽  
Vol 21 (11) ◽  
pp. 3925
Author(s):  
Ingrid Yamile Pulido ◽  
Erlide Prieto ◽  
Gilles Paul Pieffet ◽  
Lina Méndez ◽  
Carlos A. Jiménez-Junca
Keyword(s):  
Escherichia Coli ◽  
Molecular Dynamics ◽  
Pseudomonas Aeruginosa ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Residual Activity ◽  
Solvent Tolerance ◽  
Palm Fruit ◽  
E Coli ◽  
Dynamics Simulations

This study aimed to express heterologously the lipase LipA from Pseudomonas aeruginosa PSA01 obtained from palm fruit residues. In previous approaches, LipA was expressed in Escherichia coli fused with its signal peptide and without its disulfide bond, displaying low activity. We cloned the mature LipA with its truncated chaperone Lif in a dual plasmid and overexpressed the enzyme in two E. coli strains: the traditional BL21 (DE3) and the SHuffle® strain, engineered to produce stable cytoplasmic disulfide bonds. We evaluated the effect of the disulfide bond on LipA stability using molecular dynamics. We expressed LipA successfully under isopropyl β-d-1-thio-galactopyranoside (IPTG) and slow autoinducing conditions. The SHuffle LipA showed higher residual activity at 45 °C and a greater hyperactivation after incubation with ethanol than the enzyme produced by E. coli BL21 (DE3). Conversely, the latter was slightly more stable in methanol 50% and 60% (t½: 49.5 min and 9 min) than the SHuffle LipA (t½: 31.5 min and 7.4 min). The molecular dynamics simulations showed that removing the disulfide bond caused some regions of LipA to become less flexible and some others to become more flexible, significantly affecting the closing lid and partially exposing the active site at all times.

scholarly journals Identification of the Thioredoxin Partner of Vitamin K Epoxide Reductase in Mycobacterial Disulfide Bond Formation

2018 ◽  
Vol 200 (16) ◽  
Author(s):  
Na Ke ◽  
Cristina Landeta ◽  
Xiaoyun Wang ◽  
Dana Boyd ◽  
Markus Eser ◽  
...  
Keyword(s):  
Vitamin K ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Cell Envelope ◽  
Bond Formation ◽  
Disulfide Bond Formation ◽  
Vitamin K Epoxide Reductase ◽  
Content Type ◽  
E Coli ◽  
Epoxide Reductase

ABSTRACTDisulfide bonds influence the stability and activity of many proteins. InEscherichia coli, the DsbA and DsbB enzymes promote disulfide bond formation. Other bacteria, including theActinobacteria, use instead of DsbB the enzyme vitamin K epoxide reductase (VKOR), whose gene is found either fused to or in the same operon as adsbA-like gene.Mycobacterium tuberculosisand other Gram-positive actinobacteria secrete many proteins with even numbers of cysteines to the cell envelope. These organisms have predicted oxidoreductases and VKOR orthologs. These findings indicate that such bacteria likely form disulfide bonds in the cell envelope. TheM. tuberculosisvkorgene complements anE. colidsbBdeletion strain, restoring the oxidation ofE. coliDsbA. While we have suggested that thedsbAgene linked to thevkorgene may express VKOR's partner in mycobacteria, others have suggested that two other extracytoplasmic oxidoreductases (DsbE or DsbF) may be catalysts of protein disulfide bond formation. However, there is no direct evidence for interactions of VKOR with either DsbA, DsbE, or DsbF. To identify the actual substrate of VKOR, we identified two additional predicted extracytoplasmic DsbA-like proteins using bioinformatics analysis of theM. tuberculosisgenome. Using the five potential DsbAs, we attempted to reconstitute disulfide bond pathways inE. coliand inMycobacterium smegmatis, a close relative ofM. tuberculosis. Our results show that onlyM. tuberculosisDsbA is oxidized by VKOR. Comparison of the properties ofdsbA- andvkor-null mutants inM. smegmatisshows parallels to the properties ofdsbmutations inE. coli.IMPORTANCEDisulfide bond formation has a great impact on bacterial pathogenicity. Thus, disulfide-bond-forming proteins represent new targets for the development of antibacterials, since the inhibition of disulfide bond formation would result in the simultaneous loss of the activity of several classes of virulence factors. Here, we identified five candidate proteins encoded by theM. tuberculosisgenome as possible substrates of theM. tuberculosisVKOR protein involved in disulfide bond formation. We then reconstituted the mycobacterial disulfide bond formation pathway inE. coliand showed that of the five candidates, onlyM. tuberculosisDsbA is efficiently oxidized by VKOR inE. coli. We also present evidence for the involvement of VKOR in DsbA oxidation inM. smegmatis.

scholarly journals Phage Display of a Biologically Active Bacillus thuringiensis Toxin

1998 ◽  
Vol 64 (8) ◽  
pp. 2995-3003 ◽  
Author(s):  
Laura M. Kasman ◽  
Andrew A. Lukowiak ◽  
Stephen F. Garczynski ◽  
Rebecca J. McNall ◽  
Phil Youngman ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Fusion Protein ◽  
Phage Display ◽  
Biologically Active ◽  
Filamentous Phage ◽  
Enzyme Linked Immunosorbent Assay ◽  
Expression Systems ◽  
E Coli ◽  
Molar Basis ◽  
Insecticidal Toxins

ABSTRACT Activated forms of Bacillus thuringiensisinsecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensistoxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning.

scholarly journals Optimised production of disulfide-bonded fungal effectors in E. coli using CyDisCo and FunCyDisCo co-expression approaches

2021 ◽  
Author(s):  
Daniel S Yu ◽  
Megan A Outram ◽  
Emma Crean ◽  
Ashley Smith ◽  
Yi-Chang Sung ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Fungal Pathogens ◽  
Sequence Similarity ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Efficient Production ◽  
E Coli ◽  
Functional Studies ◽  
Folded Structure

Effectors are a key part of the arsenal of plant pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognised by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterise effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a co-expression system within the heterologous host E. coli termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalised co-expression approach termed FunCyDisCo (Fungi-CyDisCo) can significantly improve the production yields of numerous disulfide bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallisation of several of these reported fungal effectors. We suggest this approach could be useful when investigating the function and recognition of a broad range of disulfide-bond containing effectors.

scholarly journals DESIGN AND MOLECULAR DOCKING STUDIES OF NOVEL ANTIMICROBIAL PEPTIDES USING AUTODOCK MOLECULAR DOCKING SOFTWARE

2017 ◽  
Vol 10 (16) ◽  
pp. 28 ◽  
Author(s):  
Kiranpreet Kaur ◽  
Paranjeet Kaur ◽  
Amit Mittal ◽  
Surendra Kumar Nayak ◽  
Gopal L Khatik
Keyword(s):  
Amino Acids ◽  
Molecular Docking ◽  
Antimicrobial Peptides ◽  
Binding Affinity ◽  
Docking Studies ◽  
Molecular Structures ◽  
Short Chain ◽  
Receptor Protein ◽  
Autodock Vina ◽  
E Coli

Objective: Design of novel antimicrobial peptides and study through the molecular docking.Methods: The molecular structures were drawn in ChemBiodraw ultra and by the help of ChemBiodraw 3D, all structures were energy minimized by theMM2 method and converted to pdbextension file which is readable at the ADT interface. The AutoDock Vina (ADT) 1.5.6 software is used for molecular docking purposes.Results: Eight antimicrobial peptides (AMPs) were designed based on theMP196antimicrobial peptide. Among these KP_03R (FWRWRW-NH2) showed good binding affinity. These peptides also showed the stereochemical influence on affinity toward the3vma protein of E. coli, where AMP with R stereochemistry showed better activity than its opposite stereochemistry.  Conclusion: Novel AMPs were designed by modifications on the MP196 a short chain of amino acids antimicrobial peptides. Molecular docking software was used to determine the binding affinity between drug and receptor protein. Among all the designed peptides KP_03R(FWRWRW-NH2) showed the maximum binding affinity against thepenicillin-binding protein of E.coli and also exhibited stereoselective activity.

Sign in / Sign up

Export Citation Format

Share Document