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β-galactosidase Using Gel-Filtration Chromatography
Published in Maik W. Jornitz, Filtration and Purification in the Biopharmaceutical Industry, 2019
In order to determine the extent to which E. coli-PAD overexpresses β-galactosidase, equivalent numbers of permeabilized cells from an overnight culture of E. coli-PAD and a wild-type E. coli strain were assayed for enzyme activity. Because β-galactosidase is an inducible enzyme, its synthesis needed to be induced in the wild-type strain thus, IPTG (isopropyl-l-thio-β-D-galactoside) was added to the culture at a final concentration of 1.0 mM. IPTG, unlike lactose, is non-metabolizable and acts directly by inhibiting the lac repressor. It is the generally preferred inducer for molecular studies involving the regulation and control of the lac operon. By contrast, E. coli-PAD requires no induction because the production of β-galactosidase, driven by plasmid pRS415, is constitutive.
Protein Expression Methods
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
In a T7 expression system where the DE3 gene is under the control of a lac operon, expression is induced through the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG). Induction occurs as a result of IPTG binding to the Lac repressor protein and inducing a conformational change that prevents it from binding to the Lac promoter (Figure 5.2). Once the Lac repressor protein is removed, the E. coli strain makes mRNA for the T7 RNA polymerase using E. coli RNA polymerase, which in turn produces mRNA for the protein of interest.
Cost-effective, high-yield production of Pyrobaculum calidifontis DNA polymerase for PCR application
Published in Preparative Biochemistry & Biotechnology, 2023
Kashif Maseh, Syed Farhat Ali, Shazeel Ahmad, Naeem Rashid
A colony of E. coli BL21 CodonPlus (DE3) RIL cells, transformed with recombinant plasmid Pca-pol-pET28a, was inoculated in 20 mL LB broth (containing 50 µg/mL kanamycin) and incubated overnight at 37 °C with shaking at 200 rpm in an orbital shaker. 1% of the overnight grown culture was used to initiate 50 mL secondary culture for optimization of gene expression. IPTG concentration was optimized in a range of 0.1–1 mM. To analyze the effect of growth phase on gene expression, induction was done at culture OD600 of 0.2, 0.4, 0.6, and 0.8 and an uninduced culture was used as control. Post-induction time was optimized by taking a culture sample at one hour interval (after IPTG addition) up to five hours. Effect of growth temperature on gene expression was also studied by carrying out gene expression at 20, 30, and 37 °C. All experiments were done in triplicates.
Optimization of the 503 antigen induction strategy of Leishmania infantum chagasi expressed in Escherichia coli M15
Published in Preparative Biochemistry and Biotechnology, 2018
Luan Tales Costa de Paiva Vasconcelos, Marcos Antônio Oliveira Filho, Vitor Troccoli Ribeiro, Jaciara Silva de Araújo, Francisco Canindé de Sousa Junior, Daniella Regina Arantes Martins, Everaldo Silvino dos Santos
In this context, the isopropyl-β-d-thiogalactopyranoside (IPTG) is being used as an inducer in the expression of recombinant proteins.[8–11] Nevertheless, the fact that IPTG has a high cost and is potentially toxic to cells restricts its applications as an inducer of recombinant proteins on an industrial scale. Lactose, on the other hand, is a lower cost inducer, natural and biodegradable, which can also be used in lac operon based expression systems.[12,13] Therefore, the present study aims to analyze the influence of the IPTG and lactose as inducers for the 503 antigen expression, a protein that exhibits 100% identity with the elongation fator 1-γ of Leishmania infantum,[6] by using different concentrations and culture conditions at the time of induction.
Enhancement of prodigiosin synthetase (PigC) production from recombinant Escherichia coli through optimization of induction strategy and media
Published in Preparative Biochemistry and Biotechnology, 2018
Zhongyu You, Suping Zhang, Xiaoxia Liu, Yujie Wang
Isopropy-β-d-thiogalactoside is a common and efficient synthetic inducer for the overexpression of heterologous proteins in E. coli.[21] High concentrations of IPTG are known to inhibit cell growth.[24,25] However, 0.01 mM of IPTG is a relatively low concentration, and it was not expected to cause growth inhibition. The relatively low optimal IPTG concentration found in this study was possibly due to the expression rate of the recombinant protein and the protein folding process. As an inducer, the main function of IPTG is to combine with a repressor protein when added to the medium, so that the transcription of a foreign gene can continue.[12] Therefore, within a certain range, an increase in IPTG concentration will increase the rate of expression. Although the expression of PigC was faster when concentrations of IPTG were greater than 0.01 mM, the protein was misfolded, forming inclusion bodies. Similar results have been observed for pullulanase expression using IPTG.[10]