Purification and Characterization of Recombinant sTRAIL Expressed in Escherichia coli

2004 ◽  
Vol 36 (2) ◽  
pp. 118-122 ◽  
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.

Expression, Purification and Characterization of Amantadine Receptor in Escherichia coli

2012 ◽  
Vol 161 ◽  
pp. 88-93 ◽  
Author(s):  
Hai Xin Sun ◽  
Li Min Cao ◽  
Hong Lin ◽  
Fang Lv
Keyword(s):  
Inclusion Bodies ◽  
Equilibrium Dialysis ◽  
Molecular Size ◽  
M2 Protein ◽  
Purification And Characterization ◽  
E Coli ◽  
His Tag ◽  
Sds Page ◽  
Fast Flow

In order to obtain large quantities of broadly selective receptor as one diagnose agent to detect amantadine residue, the M2 protein gene with a His-tag was ligated into pET11a and transferred into E. coli BL21 (DE3) cell. The recombinant E. coli was cultured in liquid LB culture. SDS-PAGE result showed the recombinant M2 protein (rM2) was expressed as insoluble inclusion bodies with about 18KDa in molecular size. rM2 protein was further recognized by Western blot and purified by Ni Sepharose 6 Fast Flow and then refolded. The equilibrium dialysis result showed the rM2 protein had the binding constant of 1.1×105, and stoichiometry of 4.2. The above result showed the rM2 has the potential as biological diagnose agent to the detection of amantadine residue.

Refolding, purification, and characterization of constitutive-active human-Smad8 produced as inclusion bodies in ClearColi® BL21 (DE3)

2021 ◽  
Vol 184 ◽  
pp. 105878
Author(s):  
Carla Lizbeth Segovia-Trinidad ◽  
Bastian Quaas ◽  
Zhaopeng Li ◽  
Antonina Lavrentieva ◽  
Yvonne Roger ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Purification And Characterization

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

2005 ◽  
Vol 37 (4) ◽  
pp. 265-269 ◽  
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.

scholarly journals Expression and Isolation of N-Terminal Truncated Human Recombinant Renalase in Prokaryotic Cells

2021 ◽  
Vol 4 (3) ◽  
pp. e00158
Author(s):  
V.I. Fedchenko ◽  
A.A. Kaloshin ◽  
S.A. Kaloshina ◽  
A.E. Medvedev
Keyword(s):  
Recombinant Protein ◽  
Signal Peptide ◽  
Extracellular Space ◽  
Inclusion Bodies ◽  
High Concentration ◽  
Genetic Construct ◽  
E Coli ◽  
Insoluble Form ◽  
Guanidine Chloride ◽  
Catalytic Functions

Renalase (RNLS) is a flavoproteinin which its N-terminal peptide (residues 1-17) has several important functions. In cells, it participates in the formation of the so-called Rossmanfold (residues 2-35), needed for «accommodation» of the FAD cofactor and for performing the catalytic functions of RNLS as a FAD-dependent oxidoreductase (EC 1.6.3.5). RNLS secretion into the extracellular space is accompanied by cleavage of this peptide. The resultant truncated extracellular RNLS cannot bind FAD and therefore performs various noncatalytic functions. In this work, we have performed expression the genetic construct encoding RNLS lacking its N-terminal signal peptide (tRNLS) in E. coli Rosetta (DE3) cells. The recombinant protein was accumulated in inclusion bodies in an insoluble form, which could be solubilized in the presence of a high concentration of urea or guanidine chloride. In contrast to full-length RNLS, which was effectively solubilized in the presence of 8 M urea, tRNLS was preferentially solubilized in the presence of 6 M guanidine chloride.

Optimizing a production strategy for a nonspecific nuclease from Yersinia enterocolitica subsp. palearctica in genetically engineered Escherichia coli

2019 ◽  
Vol 366 (24) ◽  
Author(s):  
Yan Ge ◽  
Senlin Guo ◽  
Tao Liu ◽  
Chen Zhao ◽  
Duanhua Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Yersinia Enterocolitica ◽  
Inclusion Bodies ◽  
Genetically Engineered ◽  
Biological Research ◽  
Dilution Method ◽  
E Coli ◽  
Protein Renaturation ◽  
Engineered Escherichia Coli ◽  
Pharmaceutical Production

ABSTRACT A nuclease from Yersinia enterocolitica subsp. palearctica (Nucyep) is a newly found thermostable nonspecific nuclease. The heat-resisting ability of this nuclease would be extremely useful in biological research or pharmaceutical production. However, the application of this nuclease is limited because of its poor yield. This research aimed to improve Nucyep productivity by producing a novel genetically engineered Escherichia coli and optimizing the production procedures. After 4 h of induction by lactose, the new genetically engineered E. coli can express a substantial amount of Nucyep in the form of inclusion bodies. The yield was approximately 0.3 g of inclusion bodies in 1 g of bacterial pellets. The inclusion bodies were extracted by sonication and solubilized in an 8 M urea buffer. Protein renaturation was successfully achieved by dilution method. Pure enzyme was obtained after subjecting the protein solution to anion exchange. The Nucyep showed its nonspecific and heat resistant properties as previously reported (Boissinot et  al. 2016). Through a quantification method, its activity was determined to be 1.3 × 10 6 Kunitz units (K.U.)/mg. These results can serve as a reference for increasing Nucyep production.

Expression, purification and characterization of human urodilatin in E. coli

2007 ◽  
Vol 55 (2) ◽  
pp. 312-318 ◽  
Author(s):  
Ziyong Sun ◽  
Wei Lu ◽  
Yanchun Tang ◽  
Jing Zhang ◽  
Junyong Chen ◽  
...  
Keyword(s):  
Purification And Characterization ◽  
E Coli

Expression, purification and characterization of a full-length recombinant HIV-1 Vpu from inclusion bodies

2016 ◽  
Vol 128 ◽  
pp. 109-114 ◽  
Author(s):  
Zikhona Njengele ◽  
Ronel Kleynhans ◽  
Yasien Sayed ◽  
Salerwe Mosebi
Keyword(s):  
Inclusion Bodies ◽  
Full Length ◽  
Purification And Characterization ◽  
Hiv 1
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