Coding sequences of Marwari horse mature IL-4 and IL-10 gene [16 ] were subcloned into Sac I and Hind III (Thermo Scientific FastDigest, UK) restricted prokaryotic expression vector pQE30 (Qiagen, Germany). The recombinant pQE30 vectors with respective IL-4 and IL-10 insert were transformed in Escherichia coli M15 competent cells prepared by Z-competent™ E. coli transformation kit (Zymo Research, USA). Positive clones were selected on Luria Bertani agar (LBA) plates supplemented with kanamycin (30 μg/ml) and ampicillin (50 μg/ml) (HiMedia, India) and screened by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, 10 colonies were randomly picked and were grown in 5 ml of LB broth at 37°C at 170 rpm. At mid-log phase (approximate O.D600 0.6), 1 mM isopropyl-β-D-thiogalactoside (IPTG) (Promega, USA) was added to culture and was further incubated for 3-6 h. Samples from bacterial cells were prepared as per standard protocol [17 ] and run on 12% SDS-PAGE. The gel was stained by Coomassie brilliant blue dye and protein band was visualized by destaining. For further confirmation of insert, two positive recombinant clones were sequenced by Sanger method (Eurofins genomics, India) and sequences were verified by NCBI-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi ) program.
Isopropyl β d thiogalactoside iptg
Manufactured by Promega
Sourced in United States
Isopropyl-β-D-thiogalactoside (IPTG) is a synthetic analog of lactose. It is commonly used in molecular biology as an inducer of gene expression in bacterial systems, particularly in the lac operon.
Automatically generated - may contain errors
Lab products found in correlation
Pqe30, by Qiagen (1 mentions)
Z competent e coli transformation kit, by Zymo Research (1 mentions)
Ampicillin, by HiMedia (1 mentions)
Kanamycin, by HiMedia (1 mentions)
Sanger method, by Eurofins (1 mentions)
Ampicillin, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Serva Electrophoresis (1 mentions)
Maxq 4000, by Thermo Fisher Scientific (1 mentions)
4 protocols using isopropyl β d thiogalactoside iptg
1
Recombinant Expression of Marwari Horse IL-4 and IL-10
2
Recombinant HSP40 Protein Production
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The pET30a-HSP40 plasmid was constructed using the same method and primers used to construct the pVAX1-HSP40 plasmid above. This pET30a-HSP40 plasmid was transfected into Escherichia coli BL21 competent cells by heat shock at 42 °C for 1.5 min, and was induced to express the rHSP40 protein in vitro using isopropyl-β-D-thiogalactoside (IPTG, Promega, Fitchburg, Wisconsin, USA). The products with 6-His tag were purified by Ni-NTA His Bind® Resin (Novagen, Madison, Wisconsin, USA). The purified rHSP40 protein was used to coat the wells of the 96-well plates in the enzyme-linked immunosorbent assay (ELISA) to measure the concentrations of specific anti-rHSP40 antibodies.
3
Metabolite Production in E. coli
4
Recombinant Protein Expression in E. coli
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The plasmid pGEX-EG95NC -was transformed into E. coli BL21(DE3). Culture of recombinant E. coli was performed in shake flasks. In order to provide the optimum conditions for aeration and mixing, the culture volume represented 20% of the total flask volume. Luria broth medium (LB) containing 100 µg/ml of ampicillin (Serva) was inoculated with the recombinant E. coli and incubated in a shaker incubator (MaxQ 4000, ThermoScientific) at 37°C, 200 rpm for 14 h. Cell density was measured until the OD (595nm) reached approximately 0.8-1 unit and dilution made for the second passage at OD 0.05 in LB medium containing ampicillin 100 µg/ml, followed by incubated with shaking.
Protein expression was induced by addition of isopropyl-β-D-thiogalactoside (IPTG, Promega) at a concentration of 0.5 mM. Assessment of levels of recombinant protein expression was performed for 5h every hour during cultivation, E. coli cell suspensions were centrifuged at 3500g (1-15PK; SIGMA). The culture supernatant was discarded and bacterial pellets suspended in loading buffer (Tris 50 mM, Glycine 200 mM, SDS 0.1 %, beta mercaptoethanol 5%, pH 8.3) and lysed in a water bath at 95°C for 5min before chilling on ice for SDS-PAGE analysis (Fig. 3).
Protein expression was induced by addition of isopropyl-β-D-thiogalactoside (IPTG, Promega) at a concentration of 0.5 mM. Assessment of levels of recombinant protein expression was performed for 5h every hour during cultivation, E. coli cell suspensions were centrifuged at 3500g (1-15PK; SIGMA). The culture supernatant was discarded and bacterial pellets suspended in loading buffer (Tris 50 mM, Glycine 200 mM, SDS 0.1 %, beta mercaptoethanol 5%, pH 8.3) and lysed in a water bath at 95°C for 5min before chilling on ice for SDS-PAGE analysis (Fig. 3).
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