A PCR product encoding 50 aa AP2 domain and 50 aa flanking coding region was inserted into the pET‐GST vector. Recombinant proteins were expressed in E. coli BL21(DE3) strain (Invitrogen, #C600003). The transformed bacteria were inoculated in LB medium supplemented with 100 μg/ml of ampicillin (Sangon Biotech, #A100339) and incubated overnight at 37°C with shaking (200 rpm). Next, an inoculum (1:50) was made in a fresh medium and culture was incubated at 37°C with shaking (200 rpm). Until the culture reached an optical density (OD600) of 0.4 to 0.6, isopropyl‐β‐d‐thiogalactoside (Sangon Biotech, #A600168) was added to a final concentration of 0.2 mM. The culture was induced at 16°C overnight and affinity purified using glutathione resin (Thermo Fisher, #16100) according to the manufacturer’s instructions. The efficiency of protein purification was estimated by SDS–PAGE and Coomassie blue staining.
Isopropyl β d thiogalactoside
Manufactured by Sangon
Sourced in China
Isopropyl‐β‐d‐thiogalactoside is a synthetic analogue of lactose. It is commonly used as an inducer in bacterial expression systems to promote the expression of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ampicillin, by Sangon (3 mentions)
E coli bl21 de3 strain, by Thermo Fisher Scientific (1 mentions)
Glutathione resin, by Thermo Fisher Scientific (1 mentions)
Restriction enzyme, by Takara Bio (1 mentions)
Plasmid extraction kit, by Takara Bio (1 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (1 mentions)
Yeast powder, by Sangon (1 mentions)
Peptone, by Sangon (1 mentions)
Kanamycin, by Sangon (1 mentions)
Bca protein concentration determination kit, by Solarbio (1 mentions)
Dna purification kit, by Takara Bio (1 mentions)
Taq master mix, by Novoprotein (1 mentions)
Plasmid mini preparation kit, by Takara Bio (1 mentions)
Plasmid purification kit, by Takara Bio (1 mentions)
3 protocols using isopropyl β d thiogalactoside
1
Bacterial Expression and Purification of AP2 Domain
2
Controlled Error-prone PCR for Mutagenesis
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Restriction enzymes, plasmid extraction kit, and fragment purification kit were purchased from TAKARA (Dalian, China); Controlled Error-prone PCR Kit was purchased from TIANDZ (Beijing, China); ClonExpress II One Step Cloning Kit was purchased from Vazyme (Nanjing, China); yeast powder, peptone, Kanamycin, Ampicillin, Isopropyl-β-d-thiogalactoside, and l -aspartic acid were purchased from Sangon (Shanghai, China); 1,2-Diacetylbenzene was from Sigma-Aldrich (St. Louis, MO, USA). All of the chemicals used were of analytical grade.
3
Enzymatic Synthesis and Analysis
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Phenylpyruvate, p-hydroxyphenylpyruvate and nicotinamide adenine dinucleotide (NADH) were purchased from Aladdin (Shanghai, China), benzoyl formic acid was purchased from Urchem (Shanghai, China), isopropyl-β-D-thiogalactoside (IPTG) and ampicillin were purchased from Sangon Biotech (Shanghai, China). 3,4dihydroxyphenylpyruvate was synthesized according to the method described in the literature (Bai et al., 2014) .
FastPfu PCR SuperMix was purchased from Miozyme (Shanghai, China), Taq Master Mix was purchased from Novoprotein (Suzhou, China), and ABclonal MultiF Seamless Assembly was purchased from abclonal (Wuhan, China). BCA protein concentration determination kit was obtained from Solarbio (Beijing, China). Restriction endonucleases (SacⅠ, BamHI), QuickCut TM DpnⅠ, plasmid mini preparation kit, Plasmid Purification Kit and DNA purification kit were purchased from TaKaRa (Otsu, Japan). Color pre-stained standard protein Marker was purchased from Adamas (Shanghai, China). All of the above chemical reagents are analytical grade.
FastPfu PCR SuperMix was purchased from Miozyme (Shanghai, China), Taq Master Mix was purchased from Novoprotein (Suzhou, China), and ABclonal MultiF Seamless Assembly was purchased from abclonal (Wuhan, China). BCA protein concentration determination kit was obtained from Solarbio (Beijing, China). Restriction endonucleases (SacⅠ, BamHI), QuickCut TM DpnⅠ, plasmid mini preparation kit, Plasmid Purification Kit and DNA purification kit were purchased from TaKaRa (Otsu, Japan). Color pre-stained standard protein Marker was purchased from Adamas (Shanghai, China). All of the above chemical reagents are analytical grade.
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