scholarly journals Cloning, expression, purification and interaction evaluation of 30 amino acids in C terminus of Clostridium perfringens toxin with its receptor Claudin-4

2019 ◽  
Vol 2 (4) ◽  
pp. 47-55
Author(s):  
Quang Kien Huynh ◽  
An Hoang Nguyen ◽  
Quynh Thi Mong Pham ◽  
Hoan Phuoc Khai Nguyen ◽  
Hieu Van Tran
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Clostridium Perfringens ◽  
Field Effect Transistors ◽  
Oral Vaccine ◽  
Soluble Form ◽  
M Cells ◽  
Gastrointestinal Infections ◽  
E Coli ◽  
C Terminus

Oral vaccine is a strategy being the most interested about treatments of gastrointestinal infections because of many great benefits outweigh conventional injection vaccines. In order to resolve the dispersion of antigens in gastrointestinal surfaces, the immunological tolerance and also be capable to stimulate immune responses effectively, M cells are targeted for antigens delivery. A number of researches reported that 30 amino acids in C terminus of Clostridium perfringens toxin (CPE30) have a high affinity to Claudin-4 receptor presenting on M cells. It is highly indispensable to produce a resource for assessing of CPE30 binding ability so cpe30 gene was cloned into the pET-gfp plasmid by two restriction enzymes BamHI and NdeI on the E. coli DH5α strain. The expression and confirmation of the fusion protein CPE30-GFP which was induced by IPTG in E. coli BL21 (DE3) strain and assessed by SDS-PAGE and Western blot with 6xHis Taq antibody demonstrated that there was the over expression of CPE30 GFP fusion protein in the cytoplasm, mainly in the soluble form. Finally, CPE30-GFP was purified which the purity was approximately 92.3%. In vitro protein interaction measurement using silicon nanowire field-effect transistors (SiNW FETs) showed that CPE30-GFP had a good binding affinity with its receptor Claudin-4 (R4). This result laid the groundwork for the CPE30 interaction study with the M cell in vivo.

scholarly journals Cloning, expression, and purification of the M cell targeting peptide CPE16 derived from C-terminus of Clostridium perfringens enterotoxin and the binding evaluation with Claudin-r4

2019 ◽  
Vol 3 (1) ◽  
pp. 38-45
Author(s):  
Huynh Kien Quang ◽  
Mai Quoc Gia ◽  
Nguyen Hoang An ◽  
Vo Thi Thanh Ha ◽  
Tran Van Hieu
Keyword(s):  
Clostridium Perfringens ◽  
Recombinant Plasmid ◽  
Oral Vaccine ◽  
Restriction Enzymes ◽  
Soluble Form ◽  
M Cells ◽  
M Cell ◽  
Binding Ability ◽  
Clostridium Perfringens Enterotoxin ◽  
C Terminus

Developing the oral vaccine that stimulates the mucosal immune system in order to prevent the gastro-intestinal infection is an indispensable demand nowadays. Targeting the M cells, which is a sampling antigen cell, is a highly efficient solution to prevent the dispersion of antigens. Many researches demonstrate that C-terminus Clostridium perfringens enterotoxin bounds to the Claudin- 4 receptor on the M cell surface. By using bioinformatics methods, the peptide CPE16 (16 amino acid of C-terminus of Clostridium perfringens enterotoxin) was predicted to have a high affinity to Claudin-4 receptor on M cells. In this present study, CPE16-GFP was produced as a resource to assess the binding ability to M cells. Recombinant plasmid pET22b-cpe16-gfp was constructed through cloning cpe16-gfp gene into pET22b by two restriction enzymes, NdeI and XhoI, respectively. The recombinant plasmid was transformed into E. coli BL21 (DE3) strain. The expression of protein CPE16-GFP was induced by 0.5 mM IPTG and confirmed by SDS-PAGE analysis and Western blot probed with anti-6xHis antibody. CPE16-GFP protein was expressed in soluble form. CPE16- GFP was purified by using immobilized-metal affinity chromatography with the purity up to 94.14 percent. Finally, CPE16 was tested for the binding ability to recombinant GST-claudin-R4 with the use of silicon nanowire (SiNW-FET). The result showed that CPE16 interacted with GST-claudin-R4 presented by the change of the current through nanowire, compared to its counterpart control GST.  

High-Level Production of Recombinant Human Parathyroid Hormone 1-34

1998 ◽  
Vol 64 (2) ◽  
pp. 526-529 ◽  
Author(s):  
Yuji Suzuki ◽  
Masayuki Yabuta ◽  
Kazuhiro Ohsuye
Keyword(s):  
Amino Acids ◽  
Parathyroid Hormone ◽  
Fusion Protein ◽  
Human Parathyroid Hormone ◽  
E Coli ◽  
C Terminus ◽  
Secretory Type ◽  
High Level ◽  
Kex2 Protease ◽  
Level Production

ABSTRACT Expression of the synthetic human parathyroid hormone 1-34 [hPTH(1-34)] gene by a gene fusion strategy was demonstrated. hPTH(1-34) was produced at the C terminus of the partner peptides involving amino acids 1 to 97, 1 to 117, or 1 to 139 of a modifiedEscherichia coli β-galactosidase by linker peptides containing oligohistidine of different lengths. The fusion proteins in the inclusion bodies were rendered soluble with urea and subjected to site-specific cleavage with the secretory type yeast Kex2 protease. Optimal expression and enzymatic processing were achieved in the fusion protein βG-117S4HPT, constructed from amino acids 1 to 117 of β-galactosidase and the linker of HHHHPGGSVKKR. The fusion protein accumulated more than 20% of the E. coli total protein. The hPTH(1-34) was purified up to 99.5% with a good yield of 0.5 g/liter of culture. The purified product was identified as intact hPTH(1-34) by amino acid analysis and N-terminal sequencing.

Expression, Purification, and in vivo Evaluation of GFP-Fused M Cell Targeting Receptor Binding Domain of Protein FimH

2019 ◽  
Vol 26 (9) ◽  
pp. 676-683 ◽  
Author(s):  
Thanh-Hoa Nguyen-Thi ◽  
Kien-Quang Huynh ◽  
Phuong-Linh Dinh-Thi ◽  
Linh-Thuoc Tran ◽  
Yong-Suk Jang ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Oral Vaccine ◽  
Binding Domain ◽  
Soluble Form ◽  
M Cells ◽  
Receptor Binding Domain ◽  
In Vivo Evaluation ◽  
E Coli

Background: The number of oral vaccines is still limited due to many difficulties suffered in the intestinal environment, such as mucosal clearance, vast area, harsh conditions, deteriorative enzymes, impermeability, tolerance, etc. Numerous strategies have focused on directing antigen to the receptors of M cells, which is the main gateway to acquire and initiate specific responses to antigens in intestine. FimHrb is a receptor binding domain of type 1 of fimbriae from E. coli and Salmonella that can bind to GP2 receptor expressed exclusively on M cells. Objective: In this study, we evaluated the potential of FimHrb for oral vaccine development via its ability to adhere M cells. Methods: The coding gene of FimHrb fused Green Fluorescent Protein (GFP) was cloned and expressed intracellularly in E. coli host strain. The recombinant protein FimHrb-GFP was then purified by IMAC method through 6x His tag designed downstream of GFP. Finally, the purified protein was monitored its binding on murine M cells in Payer Patch region. Results: Following the methods mentioned above, the coding gene FimHrb-GFP was successfully cloned into vector pET22b and intracellularly expressed in soluble form at low temperature induction. The purity and the recovered yield of this protein were 90% and 20%, respectively. After that, the adhesion of FimHrb-GFP was monitored in murine small intestine, which showed that the protein bound to Peyer Patch region and did not restrict on M cells. Conclusion: With the present data, we revealed a candidate protein FimHrb targeted receptor on M cells for oral vaccine development and other factors in E. coli would supplement FimH to provide the specific invasion of these bacteria via M cells.

scholarly journals The Amino Acids Motif -32GSSYN36- in the Catalytic Domain of E. coli Flavorubredoxin NO Reductase Is Essential for Its Activity

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 926
Author(s):  
Maria C. Martins ◽  
Susana F. Fernandes ◽  
Bruno A. Salgueiro ◽  
Jéssica C. Soares ◽  
Célia V. Romão ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Domain ◽  
Reductase Activity ◽  
Conserved Residues ◽  
Mutant Proteins ◽  
E Coli ◽  
C Terminus ◽  
Bona Fide ◽  
Oxygen Reductase ◽  
Soluble Enzymes

Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif, -G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.

scholarly journals Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

2008 ◽  
Vol 190 (11) ◽  
pp. 4001-4016 ◽  
Author(s):  
Wallace A. Kaserer ◽  
Xiaoxu Jiang ◽  
Qiaobin Xiao ◽  
Daniel C. Scott ◽  
Matthew Bauler ◽  
...  
Keyword(s):  
Amino Acids ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Binding Motif ◽  
Inner Membrane ◽  
E Coli ◽  
Hybrid Proteins ◽  
C Terminus ◽  
N Terminus

ABSTRACT We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB + bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

scholarly journals Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.

1994 ◽  
Vol 127 (6) ◽  
pp. 1617-1626 ◽  
Author(s):  
M Furuse ◽  
M Itoh ◽  
T Hirase ◽  
A Nagafuchi ◽  
S Yonemura ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Tight Junctions ◽  
Integral Membrane Protein ◽  
Deletion Mutants ◽  
Glutathione S Transferase ◽  
E Coli ◽  
Vitro Binding ◽  
Peripheral Proteins

Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.

scholarly journals Autodisplay: Functional Display of Active β-Lactamase on the Surface of Escherichia coli by the AIDA-I Autotransporter

2000 ◽  
Vol 182 (13) ◽  
pp. 3726-3733 ◽  
Author(s):  
Claus T. Lattemann ◽  
Jochen Maurer ◽  
Elke Gerland ◽  
Thomas F. Meyer
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Surface Display ◽  
Heterologous Proteins ◽  
Bacterial Surface ◽  
E Coli ◽  
C Terminus ◽  
Protease Treatment ◽  
Efficient Expression ◽  
Cell Envelopes

ABSTRACT Members of the protein family of immunoglobulin A1 protease-like autotransporters comprise multidomain precursors consisting of a C-terminal autotransporter domain that promotes the translocation of N-terminally attached passenger domains across the cell envelopes of gram-negative bacteria. Several autotransporter domains have recently been shown to efficiently promote the export of heterologous passenger domains, opening up an effective tool for surface display of heterologous proteins. Here we report on the autotransporter domain of the Escherichia coli adhesin involved in diffuse adherence (AIDA-I), which was genetically fused to the C terminus of the periplasmic enzyme β-lactamase, leading to efficient expression of the fusion protein in E. coli. The β-lactamase moiety of the fusion protein was presented on the bacterial surface in a stable manner, and the surface-located β-lactamase was shown to be enzymatically active. Enzymatic activity was completely removed by protease treatment, indicating that surface display of β-lactamase was almost quantitative. The periplasmic domain of the outer membrane protein OmpA was not affected by externally added proteases, demonstrating that the outer membranes of E. coli cells expressing the β-lactamase AIDA-I fusion protein remained physiologically intact.

scholarly journals Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein.

2003 ◽  
Vol 50 (1) ◽  
pp. 239-247 ◽  
Author(s):  
Anna-Maria Ochocka ◽  
Marzena Czyzewska ◽  
Tadeusz Pawełczyk
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Rho Gtpase ◽  
Expression System ◽  
Cloning And Expression ◽  
Soluble Form ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Step Procedure ◽  
Shock Proteins

In this report we describe cloning and expression of human Rho GTPase activating protein (ARHGAP6) isoform 4 in Escherichia coli cells as a fusion protein with 6xHis. We cloned the ARHGAP6 cDNA into the bacterial expression vector pPROEX-1. Induction of the 6xHis-ARHGAP6 protein in BL21(DE3) and DH5alpha cells caused lysis of the cells irrespective of the kind of culture medium used. Successful expression of the fusion protein was obtained in the MC4100Deltaibp mutant strain lacking the small heat-shock proteins IbpA and IbpB. Reasonable yield was obtained when the cells were cultured in Terrific Broth + 1% glucose medium at 22 degrees C for 16 h. The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A(600) about 0.5. Under these conditions over 90% of the fusion protein was present in a soluble form. The 6xHis-ARHGAP6 protein was purified to near homogeneity by a two step procedure comprising chromatography on Ni-nitrilotriacetate and cation exchange columns. The expression system and purification procedure employed made it possible to obtain 1-2 mg of pure 6xHis-ARHGAP6 protein from 300 ml (1.5 g of cells) of E. coli culture.

scholarly journals Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP

2009 ◽  
Vol 191 (15) ◽  
pp. 4996-5009 ◽  
Author(s):  
Dirk Tischler ◽  
Dirk Eulberg ◽  
Silvia Lakner ◽  
Stefan R. Kaschabek ◽  
Willem J. H. van Berkel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Styrene Oxide ◽  
Functional Expression ◽  
Rhodococcus Opacus ◽  
Reading Frame ◽  
Arthrobacter Aurescens ◽  
C Terminus ◽  
Two Component ◽  
Rhodococcus Opacus 1Cp

ABSTRACT Sequence analysis of a 9-kb genomic fragment of the actinobacterium Rhodococcus opacus 1CP led to identification of an open reading frame encoding a novel fusion protein, StyA2B, with a putative function in styrene metabolism via styrene oxide and phenylacetic acid. Gene cluster analysis indicated that the highly related fusion proteins of Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 are involved in a similar physiological process. Whereas 413 amino acids of the N terminus of StyA2B are highly similar to those of the oxygenases of two-component styrene monooxygenases (SMOs) from pseudomonads, the residual 160 amino acids of the C terminus show significant homology to the flavin reductases of these systems. Cloning and functional expression of His10-StyA2B revealed for the first time that the fusion protein does in fact catalyze two separate reactions. Strictly NADH-dependent reduction of flavins and highly enantioselective oxygenation of styrene to (S)-styrene oxide were shown. Inhibition studies and photometric analysis of recombinant StyA2B indicated the absence of tightly bound heme and flavin cofactors in this self-sufficient monooxygenase. StyA2B oxygenates a spectrum of aromatic compounds similar to those of two-component SMOs. However, the specific activities of the flavin-reducing and styrene-oxidizing functions of StyA2B are one to two orders of magnitude lower than those of StyA/StyB from Pseudomonas sp. strain VLB120.

scholarly journals Expression of Overlapping PreS1 Fragment Recombinant Proteins for the Determination of Immunogenic Domains in HBsAg PreS1 Region

2004 ◽  
Vol 36 (6) ◽  
pp. 397-404 ◽  
Author(s):  
Wei-Guo Hu ◽  
Jun Wei ◽  
Xin-Xiu Yang ◽  
Heng-Chuan Xia ◽  
Feng Li ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Competitive Elisa ◽  
Soluble Form ◽  
Immunological Property ◽  
E Coli ◽  
C Terminus ◽  
New Vaccines ◽  
Gst Fusion Proteins

Abstract In this study, eight preS1 fragments overlapped in preS1 (21–119) region of HBV adr subtype, i.e. preS1 (21–47), preS1 (34–59), preS1 (48–70), preS1 (60–85), preS1 (71–94), preS1 (86–109), preS1 (95–119) and preS1 (21–119), were cloned by PCR, and expressed as GST fusion proteins. These GST-preS1 fusion proteins were highly expressed in soluble form in E. coli, and about 50 to 90 mg soluble fusion proteins were purified from 1 L culture. Using these fusion proteins, the immunogenic domains in preS1 (21–119) region were identified by Western blot analysis and competitive ELISA. The results showed that the immunogenic domains mainly existed in preS1 (21–59) in N-terminus and preS1 (95–109) in C-terminus, and more importantly, a major immunogenic domain preS1 (34–59), which has much stronger immunogenicity, was identified. It was also supported by the predictions of secondary structure and immunological property in the preS1 (21–119) region. The results here would be helpful for the design of new vaccines against HBV.

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