The truncated p166 capsid protein was generated from the amino acid position 452–617 of the open reading frame 2 of the following HEV strains: W01 (genotype 1, JX857689), Mexico-14 (genotype 2, M74506), US-1 (genotype 3, AF060668), and China-9829 (genotype 4, AY789225) strains, and expressed in Escherichia coli (13 (link)). Briefly, the polymerase chain reaction fragment encoding aa 452 - 617 of the HEV strains was inserted into the pET28a vectors (Novagen, Darmstadt, Germany). Then, the plasmids were used to transform competent E. coli BL21 (DE3) cells (Promega, Madison, USA). After the confirmation of the sequence of aa 452 - 617 in the plasmids by DNA sequencing, the gene expression was induced. The cells were pelleted and lysed after an incubation period and constant shaking. The suspension was clarified by centrifugation, and then the supernatant was loaded onto a column containing Ni-NTA super flow affinity resin. The column was washed, and the fusion proteins were eluted as described previously (14 (link)). The four p166 proteins were designated as p166W01, p166Mex, p166US, and p166Chn, and a mixture (p166mix) containing equal concentrations of each of the four p166 proteins was prepared.
E coli bl21 de3 cells
Manufactured by Promega
Sourced in United States
E. coli BL21 (DE3) cells are a widely used bacterial strain commonly employed in protein expression. These cells are derived from the E. coli B strain and contain the DE3 lysogen, which allows for the inducible expression of target proteins under the control of the T7 promoter system.
Automatically generated - may contain errors
Lab products found in correlation
Restriction endonucleases, by Roche (1 mentions)
Isopropyl β d thiogalactopyranoside iptg, by Roche (1 mentions)
Nickel nitrilotriacetic acid ni nta agarose, by Qiagen (1 mentions)
High fidelity dna polymerase, by Roche (1 mentions)
T4 dna ligase, by Roche (1 mentions)
Ktaprime plus, by GE Healthcare (1 mentions)
Macro prep high q resin, by Bio-Rad (1 mentions)
4 protocols using e coli bl21 de3 cells
1
Truncated HEV Capsid Protein Expression
2
Recombinant Nucleolin Protein Expression
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The pET-28a (+) vector (Novagen)
was used in E. coli BL21 (DE3) cells
(Promega) to express His-tagged recombinant nucleolin protein. Nucleolin
was purified using His-trap, Q-seph and Mono-Q columns. The His-tag
was not removed because EMSA data showed no interference with nucleolin
binding to MYC G4 before and after cleavage.
was used in E. coli BL21 (DE3) cells
(Promega) to express His-tagged recombinant nucleolin protein. Nucleolin
was purified using His-trap, Q-seph and Mono-Q columns. The His-tag
was not removed because EMSA data showed no interference with nucleolin
binding to MYC G4 before and after cleavage.
3
Engineered HEV ORF2 Capsid Mutants
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Plasmid pET-28a (+)/p179 containing the 439–617aa region of HEV ORF2 of genotype 4 HEV strain has been constructed previously in our laboratory14 (link),28 (link). The p179 mutants with wild-type asparagine (N) replaced by the cyclic aa (P) and aromatic aa (Y) at position 562 were previously designed and successfully expressed in P. pastoris14 (link). HEV capsid protein-specific mAb (1G10) was produced by our research group13 (link). Isopropyl-β-D-thiogalactopyranoside (IPTG), High-fidelity DNA polymerase, dNTP, T4 DNA ligase and restriction endonucleases were purchased from Roche (Germany). E. coli BL21(DE3) cells were purchased from Promega. HRP-conjugated goat anti-mouse was from KPL (Gaithersburg, MD, USA). Trypsin and DAB were purchased from Sigma–Aldrich (St. Louis, MO, USA). Plasmid and DNA recovery/purification kits were obtained from Axygen, Inc (USA). The nickel-nitrilotriacetic acid (Ni-NTA) Agarose was obtained from QIAGEN Sciences, MD, USA.
4
Purification and Kinetic Analysis of GES-5 Enzyme
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For protein expression, competent E. coli BL21(DE3) cells (PROMEGA, Madison, WI, USA) were transformed with recombinant plasmid blaGES-5/pet24a(+) as reported [20 (link)]. Protein purification was performed using an Äkta Prime plus chromatograph system (GE Healthcare Life Sciences, Freiburg, Germany). GES-5 (Uniprot ID Q09HD0) was conveniently purified in a single step using a Macro-Prep High Q resin (Bio-Rad, Hercules, CA, USA.). For the obtained protein, the KM values for the two reporter substrates, CENTA and nitrocefin were determined and found to be 524 μM and 208 μM, respectively [27 ].
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