scholarly journals Escherichia Coli Redox Enzyme Maturation Proteins, TorD and DmsD Interact with GTP as Shown by Native Page Assays

2010 ◽  
Vol 98 (3) ◽  
pp. 243a
Author(s):  
Vy A. Tran ◽  
Raymond J. Turner
Keyword(s):  
Escherichia Coli ◽  
Redox Enzyme ◽  
Native Page ◽  
Enzyme Maturation

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

2021 ◽  
Author(s):  
Nguyen Thi My Trinh ◽  
Tran Linh Thuoc ◽  
Dang Thi Phuong Thao
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Insoluble Fraction ◽  
Exchange Chromatography ◽  
Native Page ◽  
E Coli ◽  
Sds Page ◽  
Stimulating Factor

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

scholarly journals A dual functional redox enzyme maturation protein for respiratory and assimilatory nitrate reductases in bacteria

2019 ◽  
Vol 111 (6) ◽  
pp. 1592-1603 ◽  
Author(s):  
Benjamin J. Pinchbeck ◽  
Manuel J. Soriano‐Laguna ◽  
Matthew J. Sullivan ◽  
Victor M. Luque‐Almagro ◽  
Gary Rowley ◽  
...  
Keyword(s):  
Redox Enzyme ◽  
Dual Functional ◽  
Enzyme Maturation ◽  
Maturation Protein ◽  
Nitrate Reductases

scholarly journals ‘Come into the fold’: A comparative analysis of bacterial redox enzyme maturation protein members of the NarJ subfamily

2014 ◽  
Vol 1838 (12) ◽  
pp. 2971-2984 ◽  
Author(s):  
Catherine S. Chan ◽  
Denice C. Bay ◽  
Thorin G.H. Leach ◽  
Tara M.L. Winstone ◽  
Lalita Kuzniatsova ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Redox Enzyme ◽  
Enzyme Maturation ◽  
Maturation Protein

scholarly journals Expression of a Gene Encoding the Carrot HSP17.7 in Escherichia coli Enhances Cell Viability and Protein Solubility Under Heat Stress

HortScience ◽  
2009 ◽  
Vol 44 (3) ◽  
pp. 866-869 ◽  
Author(s):  
Hyesoon Kim ◽  
Yeh-Jin Ahn
Keyword(s):  
Escherichia Coli ◽  
Heat Stress ◽  
Protein Solubility ◽  
Small Heat Shock Protein ◽  
Immunoblot Analysis ◽  
Transformed Cells ◽  
Bacterial Cells ◽  
Native Page ◽  
E Coli ◽  
Inducible Protein

DcHSP17.7, a small heat shock protein from carrot (Daucus carota L.), was expressed in Escherichia coli to examine its functional mechanism under heat stress. When transformed cells expressing DcHSP17.7 were exposed to 50 °C for 1 h, the number of viable cells was ≈4-fold higher than that of control. When the amount of soluble proteins was compared, it was more than twofold higher in transformed cells expressing DcHSP17.7 than that in control, suggesting that DcHSP17.7 may function as a molecular chaperone preventing heat-inducible protein degradation. Native-PAGE followed by immunoblot analysis showed that in transformed E. coli, DcHSP17.7 was present in an oligomeric complex, ≈300 kDa in molecular mass, on isopropyl b-D-thiogalactopyranoside treatment. However, the complex rapidly disappeared when bacterial cells were exposed to heat stress. In carrot, DcHSP17.7 was found in the similar-sized complex (≈300 kDa), but only during heat stress (40 °C), suggesting that the functional structure of DcHSP17.7 may be different in transformed E. coli from that in carrot.

scholarly journals Analysis of enzyme induction in bacteria

1975 ◽  
Vol 152 (2) ◽  
pp. 243-254 ◽  
Author(s):  
Ry Young ◽  
Hans Bremer
Keyword(s):  
Escherichia Coli ◽  
Enzyme Induction ◽  
Enzyme Maturation ◽  
Kinetics Of ◽  
Escherichia Coli B

The theoretical relations between the induced initiation and accumulation of lac mRNA and its translation are derived, taking the kinetics of repressor–operator dissociation and enzyme maturation into account. These relations are used to evaluate observed data on lac induction and to estimate a number of parameters that characterize the transcription and translation of the β-galactosidase gene in the bacterium Escherichia coli B/r growing at three different rates (0.7–2.1 doublings/h).

scholarly journals Protein Crystallography Reveals a Role for the FS0 Cluster of Escherichia coli Nitrate Reductase A (NarGHI) in Enzyme Maturation

2010 ◽  
Vol 285 (12) ◽  
pp. 8801-8807 ◽  
Author(s):  
Richard A. Rothery ◽  
Michela G. Bertero ◽  
Thomas Spreter ◽  
Nasim Bouromand ◽  
Natalie C. J. Strynadka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Protein Crystallography ◽  
Enzyme Maturation

Sequence analysis of bacterial redox enzyme maturation proteins (REMPs)

2004 ◽  
Vol 50 (4) ◽  
pp. 225-238 ◽  
Author(s):  
Raymond J Turner ◽  
Andriyka L Papish ◽  
Frank Sargent
Keyword(s):  
Nitrate Reductase ◽  
Protein Transport ◽  
Redox Enzyme ◽  
Respiratory Enzymes ◽  
Enzyme Maturation ◽  
Maturation Protein ◽  
Tat System ◽  
Folded Proteins ◽  
Sequence Relationships ◽  
Substrate Proteins

The twin-arginine protein transport (Tat) system is a remarkable molecular machine dedicated to the translocation of fully folded proteins across energy-transducing membranes. Complex cofactor-containing Tat substrates acquire their cofactors prior to export, and substrate proteins actually require to be folded before transport can proceed. Thus, it is very likely that mechanisms exist to prevent wasteful export of immature Tat substrates or to curb competition between immature and mature substrates for the transporter. Here we assess the primary sequence relationships between the accessory proteins implicated in this process during assembly of key respiratory enzymes in the model prokaryote Escherichia coli. For each respiratory enzyme studied, a redox enzyme maturation protein (REMP) was assigned. The main finding from this review was the hitherto unexpected link between the Tat-linked REMP DmsD and the nitrate reductase biosynthetic protein NarJ. The evolutionary link between Tat transport and cofactor insertion processes is discussed.Key words: Tat translocase, twin-arginine leader, hydrogenase, nitrate reductase, TMAO reductase, DMSO reductase, formate dehydrogenase, Tor, Dms, Hya, Hyb, Fdh, Nap.

Chaperones Promote Remarkable Solubilization of Salmonella enterica serovar Enteritidis Flagellin Expressed in Escherichia coli

2020 ◽  
Vol 27 (3) ◽  
pp. 210-218
Author(s):  
Bahador Bakhtiarvand ◽  
Zahra Sadeghi ◽  
Shirin Tarahomjoo ◽  
Soheila Yaghmaie
Keyword(s):  
Escherichia Coli ◽  
Molecular Chaperones ◽  
Salmonella Enterica ◽  
Inclusion Bodies ◽  
Vaccine Development ◽  
Limited Proteolysis ◽  
Soluble Form ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Native Page ◽  
Chemical Chaperones

Background: Flagellin of Salmonella enterica serovar Enteritidis (SEF) stimulates immune responses to both itself and coapplied antigens. It is therefore used in vaccine development and immunotherapy. Removal of pathogenic S. enterica ser. Enteritidis from SEF production process is advantageous due to the process safety improvement. The protein solubility analysis using SDS-PAGE indicated that 53.49% of SEF expressed in Escherichia coli formed inclusion bodies. However, the protein recovery from inclusion bodies requires a complex process with a low yield. Objective: We thus aim to study possibility of enhancing SEF expression in E. coli in soluble form using chemical and molecular chaperones. Methods: Chemical chaperones including arginine, sorbitol, trehalose, sodium chloride and benzyl alcohol were used as cultivation medium additives during SEF expression. SEF solubilization by coexpression of molecular chaperones DnaK, DnaJ, and GrpE was also investigated. Results: All of the chemical chaperones were effective in improving SEF solubility. However, sorbitol showed the most profound effect. SEF solubilization by molecular chaperones was slightly better than that using sorbitol and this approach enhanced noticeably SEF soluble concentration and SEF solubility percentage to almost two folds and 96.37% respectively. Results of limited proteolysis assay and native PAGE indicated similar conformational states and proper folding for SEF obtained without using chaperones and for those obtained using sorbitol and the molecular chaperones. However, the molecular chaperones based system was less costly than the sorbitol based system. Conclusion: The coexpression of molecular chaperones was then considered as the most appropriate approach for soluble SEF production. Therefore, SEF production for medical purposes is expected to be facilitated.

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