scholarly journals In vivo and in vitro folding of a recombinant metalloenzyme, phosphomannose isomerase

1996 ◽  
Vol 318 (2) ◽  
pp. 437-442 ◽  
Author(s):  
Amanda E. I. PROUDFOOT ◽  
Laurence GOFFIN ◽  
Mark A PAYTON ◽  
Timothy N. C. WELLS ◽  
Alain R BERNARD
Keyword(s):  
Inclusion Bodies ◽  
Guanidine Hydrochloride ◽  
Expression Level ◽  
Phosphomannose Isomerase ◽  
Independent Manner ◽  
Body Protein ◽  
Fourfold Increase ◽  
E Coli

Phosphomannose isomerase (PMI) catalyses the interconversion of mannose 6-phosphate and fructose 6-phosphate in prokaryotic and eukaryotic cells. The enzyme is a metalloenzyme which contains 1 mol of zinc per mol of enzyme. Heterologous expression of the cDNA coding for the Candida albicans enzyme in the prokaryotic host Escherichia coli results in an expression level of up to 30% of total E. coli protein. Ten percent of recombinant PMI is expressed in the soluble fraction and 90% in inclusion bodies. Inclusion of a high level of zinc in the fermentation medium resulted in a fourfold increase in soluble protein. Co-expression of the bacterial chaperones, GroES and GroEL, resulted in a proportional twofold increase in soluble PMI while causing an overall decrease in the PMI expression level. Folding denatured PMI in vitro required reductant and zinc ions. The yield of renatured protein was increased by folding in the presence of GroEL and DnaK in an ATP-independent manner. The refolding yield of denatured soluble enzyme from a guanidine solution was threefold higher than that of folding monomerized inclusion body protein solubilized in guanidine hydrochloride. This suggests that a proportion of recombinant protein expressed in E. coli inclusion bodies may be irreversibly denatured.

scholarly journals Expression and Functional Analysis of the Argonaute Protein of Thermus thermophilus (TtAgo) in E. coli BL21(DE3)

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 524
Author(s):  
Jiani Xing ◽  
Lixia Ma ◽  
Xinzhen Cheng ◽  
Jinrong Ma ◽  
Ruyu Wang ◽  
...  
Keyword(s):  
Thermus Thermophilus ◽  
Extreme Environments ◽  
Host Cells ◽  
Loss Of Function ◽  
Independent Manner ◽  
E Coli ◽  
Argonaute Proteins ◽  
Shed Light

The prokaryotic Argonaute proteins (pAgos) have been reported to cleave or interfere with DNA targets in a guide-dependent or independent manner. It is often difficult to characterize pAgos in vivo due to the extreme environments favored by their hosts. In the present study, we expressed functional Thermus thermophilus pAgo (TtAgo) in E. coli BL21 (DE3) cells at 37 °C. Initial attempts to express TtAgo in BL21(DE3) cells at 37 °C failed. This was not because of TtAgo mediated general toxicity to the host cells, but instead because of TtAgo-induced loss of its expression plasmid. We employed this discovery to establish a screening system for isolating loss-of-function mutants of TtAgo. The E. colifabI gene was used to help select for full-length TtAgo loss of function mutants, as overexpression of fabI renders the cell to be resistant to the triclosan. We isolated and characterized eight mutations in TtAgo that abrogated function. The ability of TtAgo to induce loss of its expression vector in vivo at 37 °C is an unreported function that is mechanistically different from its reported in vitro activity. These results shed light on the mechanisms by which TtAgo functions as a defense against foreign DNA invasion.

scholarly journals The ripX Locus of Bacillus subtilis Encodes a Site-Specific Recombinase Involved in Proper Chromosome Partitioning

1999 ◽  
Vol 181 (19) ◽  
pp. 6053-6062 ◽  
Author(s):  
Stephen A. Sciochetti ◽  
Patrick J. Piggot ◽  
David J. Sherratt ◽  
Garry Blakely
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Holliday Junction ◽  
Independent Manner ◽  
Site Specific ◽  
E Coli ◽  
Chromosome Partitioning ◽  
A Site

ABSTRACT The Bacillus subtilis ripX gene encodes a protein that has 37 and 44% identity with the XerC and XerD site-specific recombinases of Escherichia coli. XerC and XerD are hypothesized to act in concert at the dif site to resolve dimeric chromosomes formed by recombination during replication. Cultures of ripX mutants contained a subpopulation of unequal-size cells held together in long chains. The chains included anucleate cells and cells with aberrantly dense or diffuse nucleoids, indicating a chromosome partitioning failure. This result is consistent with RipX having a role in the resolution of chromosome dimers inB. subtilis. Spores contain a single uninitiated chromosome, and analysis of germinated, outgrowing spores showed that the placement of FtsZ rings and septa is affected in ripXstrains by the first division after the initiation of germination. The introduction of a recA mutation into ripXstrains resulted in only slight modifications of the ripXphenotype, suggesting that chromosome dimers can form in a RecA-independent manner in B. subtilis. In addition to RipX, the CodV protein of B. subtilis shows extensive similarity to XerC and XerD. The RipX and CodV proteins were shown to bind in vitro to DNA containing the E. coli dif site. Together they functioned efficiently in vitro to catalyze site-specific cleavage of an artificial Holliday junction containing adif site. Inactivation of codV alone did not cause a discernible change in phenotype, and it is speculated that RipX can substitute for CodV in vivo.

scholarly journals Expression, purification, and renaturation of a recombinant peptide-based HIV vaccine in Escherichia coli

2017 ◽  
Vol 63 (6) ◽  
pp. 493-501 ◽  
Author(s):  
Wei He ◽  
Jiayi Shu ◽  
Jian Zhang ◽  
Zhihua Liu ◽  
Jianqing Xu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Guanidine Hydrochloride ◽  
Exchange Chromatography ◽  
Affinity Column ◽  
Dna Encoding ◽  
Iptg Induction ◽  
Recombinant Peptide

To design an epitope-based vaccine for Human immunodeficiency virus (HIV), we previously predicted 20 potential HIV epitopes using bioinformatics approaches. The combination of these 20 epitopes has a theoretical coverage of 98.1% of the population for both the prevalent HIV genotypes and Chinese human leukocyte antigen DR types. To test the immunogenicity of this vaccine in vivo, a corresponding antigen needs to be prepared. To this end, we constructed a recombinant plasmid containing DNA encoding the epitopes and GPGPG spacers and a 6-His tag for verification of protein expression and ease of purification, and then transformed Escherichia coli cells with the plasmid. After IPTG induction, the recombinant protein was expressed in the form of mainly inclusion bodies. To stabilize the structure of denatured inclusion bodies for efficient purification and renaturation in vitro, we transferred the dissolved inclusion bodies from 7 mol/L guanidine hydrochloride to 8 mol/L urea. Under denaturing conditions, the vaccine protein was purified by a 3-step process including ion-exchange chromatography and affinity column, and then renatured by stepwise dialysis. Together, the above described procedures generated 43 mg of vaccine protein per litre of fermentation medium, and the final product reached approximately 95% purity. The purified protein was capable of eliciting antigen-specific T-cell responses in immunized mice.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

scholarly journals Effects of Bacterial CLPB Protein Fragments on Food Intake and PYY Secretion

Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2223
Author(s):  
Manon Dominique ◽  
Nicolas Lucas ◽  
Romain Legrand ◽  
Illona-Marie Bouleté ◽  
Christine Bôle-Feysot ◽  
...  
Keyword(s):  
Food Intake ◽  
Peptide Yy ◽  
Molecular Mimicry ◽  
Potential Effect ◽  
In Vivo Studies ◽  
Sprague Dawley ◽  
E Coli ◽  
In Vivo Effects

CLPB (Caseinolytic peptidase B) protein is a conformational mimetic of α-MSH, an anorectic hormone. Previous in vivo studies have already shown the potential effect of CLPB protein on food intake and on the production of peptide YY (PYY) by injection of E. coli wild type (WT) or E. coli ΔClpB. However, until now, no study has shown its direct effect on food intake. Furthermore, this protein can fragment naturally. Therefore, the aim of this study was (i) to evaluate the in vitro effects of CLPB fragments on PYY production; and (ii) to test the in vivo effects of a CLPB fragment sharing molecular mimicry with α-MSH (CLPB25) compared to natural fragments of the CLPB protein (CLPB96). To do that, a primary culture of intestinal mucosal cells from male Sprague–Dawley rats was incubated with proteins extracted from E. coli WT and ΔCLPB after fragmentation with trypsin or after a heat treatment of the CLPB protein. PYY secretion was measured by ELISA. CLPB fragments were analyzed by Western Blot using anti-α-MSH antibodies. In vivo effects of the CLPB protein on food intake were evaluated by intraperitoneal injections in male C57Bl/6 and ob/ob mice using the BioDAQ® system. The natural CLPB96 fragmentation increased PYY production in vitro and significantly decreased cumulative food intake from 2 h in C57Bl/6 and ob/ob mice on the contrary to CLPB25. Therefore, the anorexigenic effect of CLPB is likely the consequence of enhanced PYY secretion.

scholarly journals Hypoxia promotes the metastasis of pancreatic cancer through regulating NOX4/KDM5A-mediated histone methylation modification changes in a HIF1A-independent manner

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Hongzhen Li ◽  
Chunyan Peng ◽  
Chenhui Zhu ◽  
Shuang Nie ◽  
Xuetian Qian ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Nadph Oxidase ◽  
Western Blot ◽  
Cancer Progression ◽  
Histone Methylation ◽  
Invasion And Metastasis ◽  
Independent Manner ◽  
Nadph Oxidase 4

Abstract Background Hypoxia is a characteristic of the tumor microenvironments within pancreatic cancer (PC), which has been linked to its malignancy. Recently, hypoxia has been reported to regulate the activity of important carcinogenic pathways by changing the status of histone modification. NOX4, a member of NADPH oxidase (NOX), has been found to be activated by hypoxia and promote cancer progression in several cancers. But whether it is involved in the epigenetic changes of tumor cells induced by hypoxia is still unclear, and its biological roles in PC also need to be explored. Methods A hypoxic-related gene signature and its associated pathways in PC were identified by analyzing the pancreatic cancer gene expression data from GEO and TCGA database. Candidate downstream gene (NOX4), responding to hypoxia, was validated by RT-PCR and western blot. Then, we evaluated the relationship between NOX4 expression and clinicopathologic parameters in 56 PC patients from our center. In vitro and in vivo assays were preformed to explore the phenotype of NOX4 in PC. Immunofluorescence, western blot and chromatin immunoprecipitation assays were further applied to search for a detailed mechanism. Results We quantified hypoxia and developed a hypoxia signature, which was associated with worse prognosis and elevated malignant potential in PC. Furthermore, we found that NADPH oxidase 4 (NOX4), which was induced by hypoxia and upregulated in PC in a HIF1A-independent manner, caused inactivation of lysine demethylase 5A (KDM5A), increased the methylation modification of histone H3 and regulated the transcription of EMT-associated gene_ snail family transcriptional repressor 1 (SNAIL1). This served to promote the invasion and metastasis of PC. NOX4 deficiency repressed hypoxia-induced EMT, reduced expression of H3K4ME3 and impaired the invasion and metastasis of PC cells; however, knockdown of KDM5A reversed the poor expression of H3KEME3 induced by NOX4 deficiency, thereby promoting EMT. Conclusions This study highlights the prognostic role of hypoxia-related genes in PC and strong correlation with EMT pathway. Our results also creatively discovered that NOX4 was an essential mediator for hypoxia-induced histone methylation modification and EMT in PC cells.

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