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

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.

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High-level production of human parathyroid hormone in Bombyx mori larvae and BmN cells using recombinant baculovirus

Gene ◽

10.1016/0378-1119(95)00599-4 ◽

1995 ◽

Vol 167 (1-2) ◽

pp. 33-39 ◽

Cited By ~ 8

Author(s):

Sinnakaruppan Mathavan ◽

Vigdis T. Gautvik ◽

Erik Rokkones ◽

Ole Kristoffer Olstad ◽

B. Najma Kareem ◽

...

Keyword(s):

Parathyroid Hormone ◽

Bombyx Mori ◽

Recombinant Baculovirus ◽

Human Parathyroid Hormone ◽

High Level ◽

Level Production ◽

Bmn Cells

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

Science and Technology Development Journal - Natural Sciences ◽

10.32508/stdjns.v2i4.809 ◽

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.

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A Novel Approach for High Level Production of a Recombinant Human Parathyroid Hormone Fragment in Escherichia coli

Bio/Technology ◽

10.1038/nbt1094-1017 ◽

1994 ◽

Vol 12 (10) ◽

pp. 1017-1023 ◽

Cited By ~ 16

Author(s):

Hermann Gram ◽

Paul Ramage ◽

Klaus Memmert ◽

Rainer Gamse ◽

Hans Peter Kocher

Keyword(s):

Escherichia Coli ◽

Parathyroid Hormone ◽

Human Parathyroid Hormone ◽

Novel Approach ◽

High Level ◽

Level Production

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High-Level Production of Human Parathyroid Hormone (hPTH) by Induced Expression inSaccharomyces cerevisiae

Protein Expression and Purification ◽

10.1006/prep.1998.0912 ◽

1998 ◽

Vol 13 (3) ◽

pp. 396-402 ◽

Cited By ~ 4

Author(s):

Randi Vad ◽

Elin Moe ◽

Kirsti Saga ◽

Aina M.V. Kvinnsland ◽

Tordis B. Øyen

Keyword(s):

Parathyroid Hormone ◽

Human Parathyroid Hormone ◽

Induced Expression ◽

High Level ◽

Level Production

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

Catalysts ◽

10.3390/catal11080926 ◽

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.

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

Journal of Bacteriology ◽

10.1128/jb.00135-08 ◽

2008 ◽

Vol 190 (11) ◽

pp. 4001-4016 ◽

Cited By ~ 26

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.127.6.1617 ◽

1994 ◽

Vol 127 (6) ◽

pp. 1617-1626 ◽

Cited By ~ 635

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.

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

Journal of Bacteriology ◽

10.1128/jb.182.13.3726-3733.2000 ◽

2000 ◽

Vol 182 (13) ◽

pp. 3726-3733 ◽

Cited By ~ 79

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.

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Systematic Analysis of a Conserved Region of the Aminoglycoside 6′-N-Acetyltransferase Type Ib

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.12.3287-3292.2001 ◽

2001 ◽

Vol 45 (12) ◽

pp. 3287-3292 ◽

Cited By ~ 23

Author(s):

Ali Shmara ◽

Natalia Weinsetel ◽

Ken J. Dery ◽

Ramona Chavideh ◽

Marcelo E. Tolmasky

Keyword(s):

Amino Acids ◽

Active Form ◽

Major Change ◽

Cysteine Residue ◽

Alanine Scanning Mutagenesis ◽

Systematic Analysis ◽

C Terminus ◽

Acetyl Group ◽

Semisynthetic Derivative ◽

High Level

ABSTRACT Alanine-scanning mutagenesis was applied to the aminoglycoside 6′-N-acetyltransferase type Ib conserved motif B, and the effects of the substitutions were analyzed by measuring the MICs of kanamycin (KAN) and its semisynthetic derivative, amikacin (AMK). Several substitutions resulted in no major change in MICs. E167A and F171A resulted in derivatives that lost the ability to confer resistance to KAN and AMK. P155A, P157A, N159A, L160A, I163A, K168A, and G170A conferred intermediate levels of resistance. Y166A resulted in an enzyme derivative with a modified specificity; it conferred a high level of resistance to KAN but lost the ability to confer resistance to AMK. Although not as pronounced, the resistance profiles conferred by substitutions N159A and G170A were related to that conferred by Y166A. These phenotypes, taken together with previous results indicating that mutant F171L could not catalyze acetylation of AMK when the assays were carried out at 42°C (D. Panaite and M. Tolmasky, Plasmid 39:123–133, 1998), suggest that some motif B amino acids play a direct or indirect role in acceptor substrate specificity. MICs of AMK and KAN for cells harboring the substitution C165A were high, suggesting that the active form of the enzyme may not be a dimer formed through a disulfide bond. Furthermore, this result indicated that the acetylation reaction occurs through a direct mechanism rather than a ping-pong mechanism that includes a transient transfer of the acetyl group to a cysteine residue. Deletion of fragments at the C terminus demonstrated that up to 10 amino acids could be deleted without a loss of activity.

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