The EspR ORF was amplified from Mtb H37Rv genomic DNA and cloned in pCDNA3.1 using BamH1 and XhoI sites (pC-EspR). pET-EspR was generated by cloning EspR ORF in pET28a using BamH1 and XhoI. pMSP12::mCherry-EspR was generated by cloning EspR ORF in pMSP12::mCherry (procured from the Addgene #30169) using KpnI and HindIII sites. pET-EspR transformed E. coli BL-21λDE3 (Agilent technologies, USA) cells were used for purification of recombinant MtbEspR protein (carrying N and C terminal 6X histidine tag) upon induction with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Fermentas, USA) for 4 h at 37°C under native conditions using Talon-affinity resin (Clontech, USA) according to the manufacturer’s protocol and eluted with 200 mM imidazole containing PBS. Subsequently, rMtbEspR was dialyzed against PBS supplemented with 50 mM NaCl, and 10% glycerol. The protein concentration was measured using 1X Bradford Reagent (Bio-Rad, USA) according to the manufacturer’s instructions and the integrity of the protein was checked on 15% SDS PAGE (Supplementary Figures S1A, B
E coli bl21 λ de3
Manufactured by Agilent Technologies
Sourced in United States
The E. coli BL21 (λ DE3) is a bacterial strain commonly used in molecular biology and protein expression. It is a derivative of the E. coli B strain, engineered to facilitate the expression of recombinant proteins. The strain contains the λ DE3 lysogen, which carries the T7 RNA polymerase gene under the control of the lac UV5 promoter, allowing for inducible protein expression.
Automatically generated - may contain errors
Lab products found in correlation
Talon affinity resin, by Takara Bio (1 mentions)
Pmsp12 mcherry, by Addgene (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
Water purification system, by Merck Group (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Dna primers, by Eurofins (1 mentions)
Dna modifying enzymes, by New England Biolabs (1 mentions)
3 protocols using e coli bl21 λ de3
1
Cloning and Purification of Recombinant EspR
2
Purification of Immunotoxin D2C7
3
Protein Expression and Purification
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DNA modifying enzymes were purchased from New England Biolabs and Thermo Fisher Scientific. DNA primers were purchased from Eurofins and Sigma. E. coli BL21 (λ DE3) was purchased from Agilent Technologies. QIAquick gel extraction kit and QIAprep spin miniprep kits were purchased from Qiagen. Ancillary reagents and EDTA [2,2′,2″,2′′′-(Ethane-1,2-diyldinitrilo) tetra acetic acid] were purchased from Sigma. All other chemical reagents used were either purchased from Sigma or Fisher Scientific, used as received unless otherwise specified. Water was purified using a Merck Millipore water purification system. Protein concentrations were determined by measuring absorbance at 280 nm using a NanoDrop spectrophotometer (Thermo Scientific).
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