Overexpression of TrxA::BjMT2 (Thioredoxin::Metallothionein Type 2 from Brassica juncea) Fusion Protein in Escherichia coli and In Vitro Cleavage by TEV Protease

2011 ◽  
Vol 183-185 ◽  
pp. 947-951 ◽  
Author(s):  
Yu Zhen Song ◽  
Ping Ping Li ◽  
Ye Jie Du
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Fusion Protein ◽  
Brassica Juncea ◽  
Tev Protease ◽  
C Terminus ◽  
Mass Spectrograph ◽  
Thiol Reagent

BjMT2 cDNA clone gene isolated from Brassica juncea was constructed in pETM-20 and expressed in E.coli as a TrxA::BjMT2 fusion protein. After affinity chromatography and cleavage from the TrxA domain, pure BjMT2 protein was obtained which strongly reacted with the thiol reagent monobromobimane. The amino acid sequence determined by mass spectrograph revealed the polypeptide contains 80 amino acids and is full of 8 and 6 cysteines with CC, CXC and C-XX-C motifs clustered near -N and -C terminus, respectively.

scholarly journals Study on Cecropin B2 Production via Construct Bearing Intein Oligopeptide Cleavage Variants

Molecules ◽  
2020 ◽  
Vol 25 (4) ◽  
pp. 1005
Author(s):  
Yi-Ting Fang ◽  
Si-Yu Li ◽  
Nien-Jen Hu ◽  
Jie Yang ◽  
Jyung-Hurng Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Fusion Protein ◽  
Md Simulation ◽  
Cleavage Reaction ◽  
Chitin Binding Domain ◽  
First Order ◽  
C Terminus ◽  
Efficient Performance ◽  
Order Rate Equation

In this study, genetic engineering was applied to the overexpression of the antimicrobial peptide (AMP) cecropin B2 (cecB2). pTWIN1 vector with a chitin-binding domain (CBD) and an auto-cleavage Ssp DnaB intein (INT) was coupled to the cecB2 to form a fusion protein construct and expressed via Escherichia coli ER2566. The cecB2 was obtained via the INT cleavage reaction, which was highly related to its adjacent amino acids. Three oligopeptide cleavage variants (OCVs), i.e., GRA, CRA, and SRA, were used as the inserts located at the C-terminus of the INT to facilitate the cleavage reaction. SRA showed the most efficient performance in accelerating the INT self-cleavage reaction. In addition, in order to treat the INT as a biocatalyst, a first-order rate equation was applied to fit the INT cleavage reaction. A possible inference was proposed for the INT cleavage promotion with varied OCVs using a molecular dynamics (MD) simulation. The production and purification via the CBD-INT-SRA-cecB2 fusion protein resulted in a cecB2 yield of 58.7 mg/L with antimicrobial activity.

Expression in Escherichia coli and in vitro processing of HIV-1 p24 fusion protein

1993 ◽  
Vol 31 (2) ◽  
pp. 225-232 ◽  
Author(s):  
Ilona Marczinovits ◽  
Imre Boros ◽  
Fouad El Jarrah ◽  
György Füst ◽  
János Molnár
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Expression In Escherichia Coli ◽  
In Vitro Processing ◽  
Hiv 1

scholarly journals The C-Terminal Flexible Domain of the Heme Chaperone CcmE Is Important but Not Essential for Its Function

2003 ◽  
Vol 185 (13) ◽  
pp. 3821-3827 ◽  
Author(s):  
Elisabeth Enggist ◽  
Linda Thöny-Meyer
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Heme Binding ◽  
Membrane Anchor ◽  
Terminal Domain ◽  
Conserved Domain ◽  
C Terminus ◽  
Helical Turn ◽  
Low Activity

ABSTRACT CcmE is a heme chaperone active in the cytochrome c maturation pathway of Escherichia coli. It first binds heme covalently to strictly conserved histidine H130 and subsequently delivers it to apo-cytochrome c. The recently solved structure of soluble CcmE revealed a compact core consisting of a β-barrel and a flexible C-terminal domain with a short α-helical turn. In order to elucidate the function of this poorly conserved domain, CcmE was truncated stepwise from the C terminus. Removal of all 29 amino acids up to crucial histidine 130 did not abolish heme binding completely. For detectable transfer of heme to type c cytochromes, only one additional residue, D131, was required, and for efficient cytochrome c maturation, the seven-residue sequence 131DENYTPP137 was required. When soluble forms of CcmE were expressed in the periplasm, the C-terminal domain had to be slightly longer to allow detection of holo-CcmE. Soluble full-length CcmE had low activity in cytochrome c maturation, indicating the importance of the N-terminal membrane anchor for the in vivo function of CcmE.

scholarly journals An In Vitro Model to Investigate the Role of Helicobacter pylori in Type 2 Diabetes, Obesity, Alzheimer’s Disease and Cardiometabolic Disease

2020 ◽  
Vol 21 (21) ◽  
pp. 8369
Author(s):  
Paola Cuomo ◽  
Marina Papaianni ◽  
Clementina Sansone ◽  
Antonio Iannelli ◽  
Domenico Iannelli ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Amino Acids ◽  
Helicobacter Pylori ◽  
Bacterial Colonization ◽  
Mammalian Target Of Rapamycin ◽  
Disease Model ◽  
Cardiometabolic Disease ◽  
Isoleucine Leucine

Helicobacter pylori (Hp) is a Gram-negative bacterium colonizing the human stomach. Nuclear Magnetic Resonance (NMR) analysis of intracellular human gastric carcinoma cells (MKN-28) incubated with the Hp cell filtrate (Hpcf) displays high levels of amino acids, including the branched chain amino acids (BCAA) isoleucine, leucine, and valine. Polymerase chain reaction (PCR) Array Technology shows upregulation of mammalian Target Of Rapamycin Complex 1 (mTORC1), inflammation, and mitochondrial dysfunction. The review of literature indicates that these traits are common to type 2 diabetes, obesity, Alzheimer’s diseases, and cardiometabolic disease. Here, we demonstrate how Hp may modulate these traits. Hp induces high levels of amino acids, which, in turn, activate mTORC1, which is the complex regulating the metabolism of the host. A high level of BCAA and upregulation of mTORC1 are, thus, directly regulated by Hp. Furthermore, Hp modulates inflammation, which is functional to the persistence of chronic infection and the asymptomatic state of the host. Finally, in order to induce autophagy and sustain bacterial colonization of gastric mucosa, the Hp toxin VacA localizes within mitochondria, causing fragmentation of these organelles, depletion of ATP, and oxidative stress. In conclusion, our in vitro disease model replicates the main traits common to the above four diseases and shows how Hp may potentially manipulate them.

scholarly journals Viable Polioviruses That Encode 2A Proteins with Fluorescent Protein Tags

2009 ◽  
Vol 84 (3) ◽  
pp. 1477-1488 ◽  
Author(s):  
Natalya L. Teterina ◽  
Eric A. Levenson ◽  
Ellie Ehrenfeld
Keyword(s):  
Fusion Protein ◽  
Fluorescent Protein ◽  
Viral Rna ◽  
Progeny Virus ◽  
C Terminus ◽  
Membrane Reorganization ◽  
Infected Cells ◽  
Viral Polyprotein ◽  
Fluorescent Protein Tags

ABSTRACT The 2A proteins of the Picornaviridae enterovirus genus are small cysteine proteinases that catalyze essential cleavages in the viral polyprotein in cis and in several cellular proteins in trans. In addition, 2A has been implicated in the process of viral RNA replication, independent of its protease functions. We have generated viable polioviruses that encode 2A proteins containing fluorescent protein tag insertions at either of two sites in the 2A protein structure. Viruses containing an insertion of Discosoma sp. red fluorescent protein (DsRed) after residue 144 of 2A, near the C terminus, produced plaques only slightly smaller than wild-type (wt) virus. The polyprotein harboring the 2A-DsRed fusion protein was efficiently and accurately cleaved; fluorescent 2A proteinase retained protease activity in trans and supported translation and replication of viral RNA, both in vitro and in infected cells. Intracellular membrane reorganization to support viral RNA synthesis was indistinguishable from that induced by wt virus. Infected cells exhibited strong red fluorescence from expression of the 2A-DsRed fusion protein, and the progeny virus was stable for three to four passages, after which deletions within the DsRed coding sequence began to accumulate. Confocal microscopic imaging and analysis revealed a portion of 2A-DsRed in punctate foci concentrated in the perinuclear region that colocalized with replication protein 2C. The majority of 2A, however, was associated with an extensive structural matrix throughout the cytoplasm and was not released from infected cells permeabilized with digitonin.

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

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.

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