Recombinant GST‐fusion proteins were expressed in E. coli BL21 (DE3) (Promega, L1195) using 0.5 mM IPTG at 25°C O/N. Bacteria were lysed in NET‐N buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P‐40) containing EDTA‐free protease inhibitor mixture (Sigma‐Aldrich, P8465) and subjected to sonication. GST proteins were purified and immobilized on glutathione agarose (Thermo Scientific, 16100). HA‐TFGwtLIR or HA‐TFGmutLIR3 or positive control (supplementary Fig 5E and F) vectors were transiently transfected in HeLa cells as indicated. Then, cells were lysed with NET‐N buffer. GST pulldown assays were performed by incubating immobilized GST‐fusion proteins with protein extract derived from HeLa cells for 2 h at 4°C with gentle agitation. Unbound proteins were removed by washing the resins five times with NET‐N buffer. GST proteins were eluted from the beads with 10 mM reduced glutathione in 50 mM Tris–HCl (pH 8.0), for 30 min with gentle agitation at room temperature. The eluted samples were boiled for 5 min in sample loading buffer and separated by SDS–PAGE.
E coli bl21 de3
Manufactured by Promega
Sourced in United States
E. coli BL21 (DE3) is a bacterial strain commonly used in molecular biology and protein expression applications. It is a genetically modified derivative of the E. coli B strain, designed to facilitate the production of recombinant proteins. The strain contains the DE3 lysogen, which allows for inducible expression of T7 RNA polymerase, enabling the expression of genes under the control of the T7 promoter.
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Lab products found in correlation
Glutathione agarose, by Thermo Fisher Scientific (1 mentions)
T4 ligase, by Takara Bio (1 mentions)
Dna marker, by Takara Bio (1 mentions)
Plasmid extraction kit, by Qiagen (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Pyrobest dna polymerase, by Takara Bio (1 mentions)
Isopropyl β d 1 thiogalactopyranoside iptg, by Merck Group (1 mentions)
Dpni, by Takara Bio (1 mentions)
T4 polynucleotide kinase, by Takara Bio (1 mentions)
Miniprep kit, by Qiagen (1 mentions)
Pcr product purification kit, by Roche (1 mentions)
T4 ligase, by Roche (1 mentions)
Anti t7 tag antibody, by Abcam (1 mentions)
Pbluescript 2 ks vector, by Thermo Fisher Scientific (1 mentions)
Dna extraction kit, by Qiagen (1 mentions)
Dna polymerase, by Roche (1 mentions)
Ampicillin, by Roche (1 mentions)
Agarose gel extraction kit, by Roche (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Bl21 de3, by Promega (1 mentions)
8 protocols using e coli bl21 de3
1
GST-Fusion Protein Pulldown Assay
2
Recombinant Protein Expression in E. coli
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E. coli DH5α and E. coli BL21 (DE3) (Promega Company), used as hosts for the propagation of plasmids and recombinant proteins expression, respectively, were grown in Luria-Bertani (LB) media containing yeast extract 5 g·L-1, tryptone 10 g·L-1, NaCl 5 g·L-1 at 37°C and pH7.0 with appropriate antibiotic supplemented. T. maritima was purchased from American Type Culture Collection (ATCC 43589). The pET-20b plasmid was obtained from Novagen Co. Ltd. Restriction enzyme, Pyrobest DNA polymerase, T4 polynucleotide kinase, T4 ligase, DNA marker, and DpnI were purchased from Takara (Dalian, China) and used according to the manufacture’s instructions. Gene purification kit and plasmid extraction kit were purchased from QIAGEN Company. Ampicillin and isopropyl β-D-1-thiogalactopyranoside (IPTG) were obtained from Sigma-Aldrich Co. Ltd. All other chemicals used in the study were of analytical-grade purity from commercial sources unless mentioned otherwise.
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E. coli DH5α and E. coli BL21 (DE3) (Promega Company), used as hosts for the propagation of plasmids and recombinant proteins expression, respectively, were grown in Luria-Bertani (LB) media containing yeast extract 5 g·L-1, tryptone 10 g·L-1, NaCl 5 g·L-1 at 37°C and pH7.0 with appropriate antibiotic supplemented. T. maritima was purchased from American Type Culture Collection (ATCC 43589). The pET-20b plasmid was obtained from Novagen Co. Ltd. Restriction enzyme, Pyrobest DNA polymerase, T4 polynucleotide kinase, T4 ligase, DNA marker, and DpnI were purchased from Takara (Dalian, China) and used according to the manufacture’s instructions. Gene purification kit and plasmid extraction kit were purchased from QIAGEN Company. Ampicillin and isopropyl β-D-1-thiogalactopyranoside (IPTG) were obtained from Sigma-Aldrich Co. Ltd. All other chemicals used in the study were of analytical-grade purity from commercial sources unless mentioned otherwise.
3
Synthesis and Purification of UDP-Sugars
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UDP and UDP-Galp were purchased
from Sigma. UDP-Galf was synthesized following the
chemo-enzymatic procedure developed by the Lowary group.20 (link) Pfu Ultra Hotstart High-Fidelity DNA polymerase
was obtained from Agilent Technologies. DpnI was
from Fisher. Escherichia coli TOP-10
chemical competent cells were obtained from Invitrogen. E. coli BL21 (DE3) chemical competent cells were
from Promega (Madison, WI). The plasmid miniprep kit was from Qiagen.
Primers were from Integrated DNA Technology (IDT). All other buffers
and chemicals were purchased from Fisher Scientific.
from Sigma. UDP-Galf was synthesized following the
chemo-enzymatic procedure developed by the Lowary group.20 (link) Pfu Ultra Hotstart High-Fidelity DNA polymerase
was obtained from Agilent Technologies. DpnI was
from Fisher. Escherichia coli TOP-10
chemical competent cells were obtained from Invitrogen. E. coli BL21 (DE3) chemical competent cells were
from Promega (Madison, WI). The plasmid miniprep kit was from Qiagen.
Primers were from Integrated DNA Technology (IDT). All other buffers
and chemicals were purchased from Fisher Scientific.
4
Cloning and Expression of Rhodococcus Enzymes
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Genomic DNA was obtained from Rhodococcus erythropolis IGTS8. The cloning vector and host were pBluescript II KS (+) vector (Fermentas) and E. coli DH5a cells (Novagen). Furthermore, the expression of wild-type and mutant enzymes was done by using the pET-23a (+) (Novagen) and E. coli BL21 (DE3) (Promega). DNA polymerase, Bam HI and Eco RI, T4 Ligase, Ampicillin from Roche Diagnostic (Germany); and DNA ladders, protein markers, T4 DNA ligase and anti-T7 tag antibody were purchased from Abcam Company. NADH and FMN sodium salts, and the IPTG- X-Gal were purchased from Sigma Company. The DNA extraction kit and high pure plasmid purification kit were obtained from Qiagen Company. The agarose gel extraction kit and the PCR product purification kit were obtained from the Roche company. The LB medium containing 100 μg/ml Ampicillin was used in the benchtop operations at 37 °C.
5
Cloning and Purification of Helicobacter pylori UreC
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The gene encoding the UreC of H. pylori was amplified from the genomic DNA of H. pylori UMAB41 using a forward primer (5′-AAAACATATGAAAAAGATTAGCAGGAAAG-3′) with a NdeI cutting site (italic) and a reverse primer (5′-CCGCTCGAGCTACCGCGCCATCTTCCACCAG-3′) with a XhoI cutting site (italic). Following double digestion with NdeI and XhoI, the purified full-length gene was ligated into correspondingly double-digested pET-30a(+) (Novagen, Madison, WI). The recombinant plasmids were then transformed into competent E.coli BL21 (DE3) (Promega, Madison, WI). The transformants were grown until an OD600 of 0.6, and over expression of the cloned UreC was induced at 30°C by IPTG (1.0 mM). The E. coli cells were harvested by centrifugation, and total cell protein was isolated using the BugBuster® Protein Extraction Reagent (Novagen, Madison, WI). Purification of the overexpressed UreC was achieved using a Ni-NTA kit (Novagen, Madison, WI) per manufacturer’s instructions. The UreC protein was analyzed by SDS-PAGE and visualized after staining with Coomassie Blue R-250. The UreC protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA).
6
Expression of Mycobacterium and Escherichia Strains
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M. tuberculosis Beijing strain was from the Laboratory of Bacteriology, Puslitbang Kemenkes RI while the M. tuberculosis H37Rv strain was from the Laboratory of Tuberculosis, Microbiology Department, University of Indonesia; E. coli DH5α and the plasmid pET-28a were purchased from Novagen, USA; E. coli BL-21(DE3) was from Promega, USA.
The Beijing and H37Rv strains were grown on Lowenstein-Jensen agar at 37°C, while E.coli DH5α and BL21(DE3) cells were in Luria-Bertani (LB) broth or on LB agar in presence of kanamycin (30 µg/ml) wherever appropriate.
The Beijing and H37Rv strains were grown on Lowenstein-Jensen agar at 37°C, while E.coli DH5α and BL21(DE3) cells were in Luria-Bertani (LB) broth or on LB agar in presence of kanamycin (30 µg/ml) wherever appropriate.
7
Cloning and Purification of CsGRN
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The methods of gene cloning and purification of recombinant CsGRN were used as our previous study (Wang et al., 2017 (link)). Briefly, the ORF of CsGRN (GenBank KY855531) has a genome length of 714 bp and was cloned into the pMAL-c2x vector (preserved in our lab). Following digestion with restriction enzymes, the plasmid DNA was attached to the pET-28a (+) expression vector (Novagen, Darmstadt, Germany), before being introduced into the E. coli BL21 (DE3) (Promega). Then, we induced selected clones at 20°C for 12–18 h with 1 mM IPTG (Sigma, Guangzhou, China) following growth. By using the Amylose Resin-Bind Purification Kit (BioLabs), we purified recombinant protein (MBP-GRN) from the supernatant.
8
Alkaliphilic Bacterium Growth Conditions
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The alkaliphilic bacterium B. lehensis G1 was grown in Horikoshi Broth (HB). Escherichia coli JM109 and E. coli BL21 (DE3) (Promega, Madison, WI, USA) were used as a cloning host and expression host, respectively, in the present study. pET21a (+) from Novagen (Merck KGaA, Darmstadt, Germany) was used as an expression plasmid. E. coli strains harbouring the recombinant plasmid were grown in Luria-Bertani (LB) media supplemented, when necessary, with 100 µg/ml ampicillin.
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