EXPRESSION OF THE STAPHYLOCOCCUS AUREUS LUKE GENE IN THE ESCHERICHIA COLI BL21(DE3) STRAIN

Two-component leukotoxins are important virulence factors for Staphylococcus aureus. Despite efforts made to study S. aureus leukotoxins, the direct mechanism of action of these toxins during infection has not been determined. However, the observation that deletion of LukED significantly attenuates highly virulent S. aureus strains supports the hypothesis that selective inhibition of LukE / D may be useful in the development of new aspects of S. aureus infection control. For this purpose, this work was carried out to test the expression and obtain a recombinant form of the LukE protein in E.coli cells. The LukE gene was cloned into the pET28-c (+) / GFP vector containing the gfp gene. Two fused genes carrying a hexahistidine tag were expressed in cells of the E. coli BL21(DE3) strain. It was found that the 6His-GFP-LucE protein aggregates in inclusion bodies. 6His-GFP-LucE was washed out of inclusion bodies with high molar urea. The 6His-GFP-LucE protein was purified by metal affinity chromatography. Research results can be applied to obtain recombinant protein including strategies for inhibition of toxin activity.

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On-column Refolding of an Insoluble His6-tagged Recombinant EC-SOD Overexpressed in Escherichia coli

Acta Biochimica et Biophysica Sinica ◽

10.1111/j.1745-7270.2005.00035.x ◽

2005 ◽

Vol 37 (4) ◽

pp. 265-269 ◽

Cited By ~ 11

Author(s):

Xi-Qiang Zhu ◽

Su-Xia Li ◽

Hua-Jun He ◽

Qin-Sheng Yuan

Keyword(s):

Escherichia Coli ◽

Inclusion Bodies ◽

Mass Ratio ◽

Chain Reaction ◽

Expression Plasmid ◽

Protein Subunits ◽

E Coli ◽

Metal Affinity ◽

Polymerase Chain ◽

Escherichia Coli Expression

Abstract The EC-SOD cDNA was cloned by polymerase chain reaction (PCR) and inserted into the Escherichia coli expression plasmid pET-28a(+) and transformed into E. coli BL21(DE3). The corresponding protein that was overexpressed as a recombinant His6-tagged EC-SOD was present in the form of inactive inclusion bodies. This structure was first solubilized under denaturant conditions (8.0 M urea). Then, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione (GSH) and oxidized glutathione (GSSG). The mass ratio of GSH to GSSG was 4:1. The purified enzyme was active, showing that at least part of the protein was properly refolded. The protein was made concentrated by ultrafiltration, and then isolated using Sephacryl S-200 HR. There were two protein peaks in the A280 profile. Based on the results of electrophoresis, we concluded that the two fractions were formed by protein subunits of the same mass, and in the fraction where the molecular weight was higher, the dimer was formed through the disulfide bond between subunits. Activities were detected in the two fractions, but the activity of the dimer was much higher than that of the single monomer. The special activities of the two fractions were found to be 3475 U/mg protein and 510 U/mg protein, respectively.

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Purification and Characterization of Recombinant sTRAIL Expressed in Escherichia coli

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/36.2.118 ◽

2004 ◽

Vol 36 (2) ◽

pp. 118-122 ◽

Cited By ~ 8

Author(s):

Xiao-Xia Xia ◽

Ya-Ling Shen ◽

Dong-Zhi Wei

Keyword(s):

Inclusion Bodies ◽

Tumor Cell Line ◽

Single Step ◽

Dilution Method ◽

High Concentration ◽

Purification And Characterization ◽

E Coli ◽

Metal Affinity ◽

Soluble Trail

Abstract As a potential anti-tumor protein, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has drawn considerable attention. This report presented the purification and characterization of soluble TRAIL, expressed as inclusion bodies in E. coli. sTRAIL inclusion bodies were solubilized and refolded at a high concentration up to 0.9 g/L by a simple dilution method. Refolded protein was purified to electrophoretic homogeneity by a single-step immobilized metal affinity chromatography. The purified sTRAIL had a strong cytotoxic activity against human pancreatic tumor cell line 1990, with ED50 about 1.5 mg/L. Circular dichroism and fluorescence spectrum analysis showed that the refolded sTRAIL had a structure similar to that of native protein with β-sheet secondary structure. This efficient procedure of sTRAIL renaturation may be useful for the mass production of this therapeutically important protein.

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Evolution of Multidrug Resistance during Staphylococcus aureus Infection Involves Mutation of the Essential Two Component Regulator WalKR

PLoS Pathogens ◽

10.1371/journal.ppat.1002359 ◽

2011 ◽

Vol 7 (11) ◽

pp. e1002359 ◽

Cited By ~ 171

Author(s):

Benjamin P. Howden ◽

Christopher R. E. McEvoy ◽

David L. Allen ◽

Kyra Chua ◽

Wei Gao ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Multidrug Resistance ◽

Aureus Infection ◽

Two Component

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The SaeR/S Two Component System: The Security System of Staphylococcus aureus

10.15788/curio1 ◽

2021 ◽

Vol 1 (1) ◽

pp. 4-9

Author(s):

Owen Burroughs ◽

Keyword(s):

Staphylococcus Aureus ◽

Security System ◽

Human Immune System ◽

Two Component System ◽

Component System ◽

Aureus Infection ◽

System A ◽

Two Component ◽

Human Immune ◽

New Treatments

Staphylococcus aureus (S. aureus) is a common human pathogen that is responsible for thousands of deaths each year. The bacterium’s severity is caused, in part, by its ability to detect and evade the human immune system. In this article, Owen Burroughs, an undergraduate researcher in the lab of Dr. Jovanka Voyich, describes his research into the SaeR/S two-component system, a “security system” that allows S. aureus to avoid being killed by immune cells. Over the course of Owen’s research, the Voyich lab has determined that the proteins SaeP and SaeQ likely play a major role in the functioning of this security system. By helping us better understand the interactions between S. aureus and its host, this research could pave the way for new treatments and therapies for severe S. aureus infection.

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TmSpz6 Is Essential for Regulating the Immune Response to Escherichia coli and Staphylococcus aureus Infection in Tenebrio molitor

Insects ◽

10.3390/insects11020105 ◽

2020 ◽

Vol 11 (2) ◽

pp. 105 ◽

Cited By ~ 7

Author(s):

Tariku Tesfaye Edosa ◽

Yong Hun Jo ◽

Maryam Keshavarz ◽

Young Min Bae ◽

Dong Hyun Kim ◽

...

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Immune Response ◽

Expression Analysis ◽

Fungal Infections ◽

Specific Expression ◽

Reading Frame ◽

E Coli ◽

Aureus Infection ◽

Toll Receptor

Spätzle is an extracellular protein that activates the Toll receptor during embryogenesis and immune responses in Drosophila. However, the functions of the spätzle proteins in the innate immune response against bacteria or fungi in T. molitor are not well understood. Therefore, in this study, the open reading frame (ORF) of TmSpz6 was identified and its function in the response to bacterial and fungal infections in T. molitor was investigated using RNAi. The highest expression of TmSpz6 was in prepupae, and 3- and 6-day-old pupae, while remarkable expression was also observed in other stages. The tissue-specific expression analysis showed that TmSpz6 expression was highest in the hemocytes of larvae. TmSpz6 expression was highly induced when challenged with Escherichia coli, Staphylococcus aureus, or Candida albicans at 6 h post-injection; however, TmSpz6-silenced larvae were significantly more susceptible to only E. coli and S. aureus infection. The antimicrobial peptides (AMPs) gene expression analysis results show that TmSpz6 mainly positively regulated the expression of TmTencin-2 and -3 in response to E. coli and S. aureus infection. Collectively, these results suggest that TmSpz6 plays an important role in regulating AMP expression and increases the survival of T. molitor against E. coli and S. aureus.

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The SaeR/S Two Component System: The Security System of Staphylococcus aureus

10.15788/f2021.curio1 ◽

2021 ◽

Vol 1 (1) ◽

pp. 4-9

Author(s):

Owen Burroughs ◽

◽

Jovanka Voyich ◽

Keyword(s):

Staphylococcus Aureus ◽

Security System ◽

Human Immune System ◽

Two Component System ◽

Component System ◽

Aureus Infection ◽

System A ◽

Two Component ◽

Human Immune ◽

New Treatments

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The SaeR/S two-component system induces interferon-gamma production in neutrophils during invasive Staphylococcus aureus infection

Microbes and Infection ◽

10.1016/j.micinf.2013.05.004 ◽

2013 ◽

Vol 15 (10-11) ◽

pp. 749-754 ◽

Cited By ~ 8

Author(s):

Robert L. Watkins ◽

Oliwia W. Zurek ◽

Kyler B. Pallister ◽

Jovanka M. Voyich

Keyword(s):

Staphylococcus Aureus ◽

Interferon Gamma ◽

Two Component System ◽

Component System ◽

Aureus Infection ◽

Two Component

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Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128900 ◽

1987 ◽

Vol 45 ◽

pp. 924-925

Author(s):

F. A. Durum ◽

R. G. Goldman ◽

T. J. Bolling ◽

M. F. Miller

Keyword(s):

Inclusion Bodies ◽

Large Scale ◽

Recombinant Dna ◽

Test System ◽

Cell Protein ◽

Gram Negative Bacteria ◽

Cytoplasmic Inclusions ◽

Isolation And Purification ◽

E Coli ◽

Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

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