Recombinant PbICL protein was obtained as described by Cruz [14] (link). Briefly, Pbicl cDNA was inserted into the pET-32a (+) expression vector (Novagen, Inc,). The resulting plasmid was transformed into E. coli BL21 C43 (DE3) cells, and expression was induced at an A600 of 0.6 by the addition of 1 mm (final concentration) isopropyl thio-β-D-galactoside (IPTG) (Sigma-Aldrich). After induction, the cells were incubated for another 2 h at 36°C with shaking at 200 rpm. The cells were harvested by centrifugation at 10,000×g for 5 min at 4°C and resuspended in 1 × NaCl/Pi buffer. After incubation for 30 min with 100 µg/mL lysozyme, the cells were lysed by extensive sonication. The sample was centrifuged at 4°C and 8,000×g for 15 min, and the supernatant, which contained the soluble protein fraction, was collected. His-tagged ICL was purified using the Ni-NTA Spin Kit (Qiagen), and the tags were subsequently removed by the addition of EKMax™ enterokinase (Invitrogen).
Isopropylthio β d galactoside iptg
Manufactured by Merck Group
Sourced in United States
Isopropylthio-β-d-galactoside (IPTG) is a synthetic chemical compound used in molecular biology as an inducer of gene expression. Its core function is to activate the lac operon in bacteria, which leads to the expression of desired genes. IPTG is commonly used in experiments involving recombinant protein production.
Automatically generated - may contain errors
Lab products found in correlation
Ni nta spin kit, by Qiagen (1 mentions)
Lb broth, by Merck Group (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Nitrilotriacetic acidmetal ion affinity chromatography, by GE Healthcare (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti flag m1 agarose affinity gel, by Merck Group (1 mentions)
Bradford dye binding assay, by Bio-Rad (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Endofree plasmid giga kit, by Qiagen (1 mentions)
4 methylumbelliferyl acetate, by Merck Group (1 mentions)
α naphtyl acetate, by Merck Group (1 mentions)
Phenylacetate, by Merck Group (1 mentions)
P nitrophenyl esters, by Merck Group (1 mentions)
8 protocols using isopropylthio β d galactoside iptg
1
Recombinant PbICL Protein Purification
2
GFP Expression Analysis by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In order to evaluate the GFP expression intensity, flow cytometric analysis was run. The confirmed bacterial clone was inoculated and cultured in LB broth (Merck, NJ, USA) and induced by 1 mM isopropylthio-β-D-galactoside (IPTG) (Sigma-Aldrich, MI, USA) for overnight at 37°C and 250 rpm in a shaking incubator. Cultures were precipitated and subsequently washed twice and resuspended in phosphate buffered saline (PBS) (pH = 7.4). GFP expression intensity was analyzed with an FACSCalibur flow cytometer (Becton Dickinson, NJ, USA) by accumulating up to 100,000 events per tube. The pUC57 transformed E. coli Top10F’ was used as negative control to enable correct compensation.
3
Cloning and Mutagenesis of SrtA Enzyme
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A 741 bp DNA fragment encoding the SrtA gene was amplified by polymerase chain reaction (PCR) from the S. mutans chromosome using the primer pairs SmsrtA-F/SmsrtA-R, the amplified fragment was digested with BamHI and XhoI, and cloned into the vector pET28a, yielding pET28SmsrtA. Point mutations in the gene-encoding SrtA were generated using a QuikChange site-directed mutagenesis kit (Stratagene, San Diego, CA, USA) to yield L111A-SrtA, L116A-SrtA, and R213A-SrtA. The genes of point mutations were confirmed by sequencing by Sangon Biotech Co., Ltd (Shanghai, China). To obtain recombinant wild-type and mutant SrtA proteins, Escherichia coli strain BL21 (DE3) was transformed with pET28SmsrtA and the SrtA mutant constructs. Protein expression was induced with 0.5 mM isopropylthio-β-d -galactoside (IPTG) (Sigma) at the mid-log-phase for 12 h at 16 °C, respectively. The soluble His-tagged wild-type and mutant SrtA proteins were further purified using the Ni-NTA system, as described in a previous study [13 (link)]. All primers used in this study are listed in Table 1 .
4
Recombinant Expression and Purification of CMFO Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The synthesized gene sequence encoding CMFO, based on a tandem-linked fusion of Rv2875, Rv3044, Rv2073c, and Rv0577 with a c-terminal His tag, was used to construct the recombinant prokaryotic expression plasmid pET30b-CMFO. The plasmid was used to transform Escherichia coli BL21 strains. Other recombinant plasmids expressing single multistage antigens were also constructed (Supplementary Table 1). The expressions of the multistage protein CMFO and single antigens were induced with 1 mM isopropyl thio-β-d -galactoside (IPTG; Cat#367931; Sigma, St. Louis, MO, USA) for 4 h, and nitrilotriacetic acid-metal ion affinity chromatography (Cat#29058803; GE Healthcare, Piscataway, NJ, USA) was performed to purify the recombinant proteins, as previously described (Wang et al., 2015 (link)). After dialysis against sterile phosphate-buffered saline (PBS), each recombinant protein was lyophilized and diluted in PBS, using pyrogen-free reagents. Residual endotoxin contamination in the solution was verified to be < 0.1 Endotoxin Unit/mL. Finally, the protein concentration was detected using a BCA Protein Assay Kit (Cat#P0009; Beyotime, Shanghai, China). The purified proteins were identified by 12% SDS-PAGE.
5
Recombinant BMP-2/Fc Protein Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human BMP-2 linked with the Fc fragment was obtained form the Department of Orthopedics, Shandong Provincial Hospital Affiliated to Shandong University (Jinan, China). The gene was sequenced by Invitrogen (Thermo Fisher Scientific, Inc.) and the BMP-2/Fc gene (200 ng) was inserted into pET27b vectors and named pET27b-BMP-2/Fc. The pET27b-BMP-2/Fc was expressed in Escherichia coli Rossetta (Invitrogen; Thermo Fisher Scientific, Inc.) by transforming the recombinant pET27b-BMP-2/Fc plasmid using electro transformation (40 (link)). The bacteria were grown in lysogeny broth medium (Invitrogen; Thermo Fisher Scientific, Inc.) for 12 h at 37°C and 0.5 mM isopropylthio-β-d-galactoside (IPTG, Sigma-Aldrich; Merck KGaA) was used to induce BMP-2/Fc expression. Subsequently, the cells were disrupted following a 12 h induction with IPTG and dissolved in 15 ml PBS. The protein underwent denaturation for 12 h at 90°C and renaturation for 12 h at 4°C and ion exchange chromatography was used to purify the protein of interest. The purified protein was collected and 1.5% gel filtration chromatography (glucan) further purified the rBMP-2/Fc protein.
6
Purification of FLAG-tagged CcsB Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression plasmid for CcsB pYC01 (Supplementary Table 7 ) was transformed into E. coli strain BL21 (DE3) for expression of CcsB. Luria Broth media (1 L) supplemented with ampicillin (100 mg/L) inoculated with BL21(DE3)/pYC-1 was grown to an OD600 of 0.6. Protein expression was then induced with 0.12 mM of isopropylthio-β-d-galactoside (IPTG, Sigma-Aldrich), followed by further incubation with shaking at 250 rpm at 16°C for 16 hours. All enzyme purification steps were conducted at 4°C. E. coli cells were harvested by centrifugation (3750 rpm, 15 minutes, 4°C), resuspended in 20 mL TBS buffer and lysed with sonication on ice. Cellular debris was removed by centrifugation (14000 g, 0.5 h, 4°C). FLAG-tagged proteins were purified by using ANTI-FLAG®M1 Agarose Affinity Gel (Sigma-Aldrich), following the supplied protocols. The cleared cell lysate was applied onto a gravity flow column with packed ANTI-FLAG Agarose Affinity Gel. After extensive washing steps, CcsB was eluted with the FLAG peptide elution buffer (100 μg/mL FLAG peptide, 50 mM Tris-HCl, pH 7.4, 100 mM NaCl). Purified proteins were concentrated and buffered exchanged into 50 mM potassium phosphate buffer (pH 7.0) +20% glycerol, concentrated, aliquoted and flash frozen. Protein concentrations were determined using the Bradford dye-binding assay (Biorad).
7
Recombinant DNA Vaccine Development
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The genes coding Rv3126c, Rv2626, Rv1813, Rv2031, Rv3426, Rv1996, Rv3873, Rv2028c, Rv2029c and Ag85A (Rv3804c) were amplified by PCR using Mtb H37Rv ATCC genomic DNA as a template. After digestion with EcoR I and BamH I, the PCR products were inserted into the pVAX1 plasmid. The recombinant DNA vaccines were purified with Qiagen columns (EndoFree Plasmid Giga kit, Qiagen, Germany), quantified by Nano-Drop 2000 (Thermo Fisher Scientific, USA), and then eluted in pyrogen-free deionized water to the concentration of 1 mg/mL. To obtain the encoded antigens, the recombinant plasmids were transformed into E. coli BL21 (DE3) cells, which were then induced with 1 mM isopropyl thio-β-D-galactoside (IPTG; Sigma, Germany) and disrupted via sonication. The expressed proteins were purified using Ni-nitrilotriacetic acid resin.
8
Purification and Analysis of Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isopropyl thio-β-D-galactoside (IPTG), p-Nitrophenyl esters, phenyl acetate, α-naphtyl acetate, β-naphtyl acetate, and 4-methyl umbelliferyl acetate were procured from SIGMA ALDRICH (USA). Ni-NTA resin for affinity chromatography was purchased from Qiagen, Germany. Standard proteins for size exclusion chromatography were purchased from Bio-Rad laboratories (USA). PCR primers were procured from Bio Serve, India. Other reagents and chemicals were procured from Himedia.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!