Protein Expression Prokaryotic cells - E. coli D1

Protein expression refers to the techniques in which a protein of interest is synthesized, modified or regulated in cells. The blueprints for proteins are stored in DNA which is then transcribed to produce messenger RNA (mRNA). mRNA is then translated into protein. In prokaryotes, this process of mRNA translation occurs simultaneously with mRNA transcription. In eukaryotes, these two processes occur at separate times and in separate cellular regions (transcription in nucleus and translation in the cytoplasm). Recombinant protein expression utilizes cellular machinery to generate proteins, instead of chemical synthesis of proteins as it is very complex. Proteins produced from such DNA templates are called recombinant proteins and DNA templates are simple to construct. Recombinant protein expression involves transfecting cells with a DNA vector that contains the template. The cultured cells can then transcribe and translate the desired protein. The cells can be lysed to extract the expressed protein for subsequent purification. Both prokaryotic and eukaryotic protein expression systems are widely used. The selection of the system depends on the type of protein, the requirements for functional activity and the desired yield. These expression systems include mammalian, insect, yeast, bacterial, algal and cell-free. Each of these has pros and cons. Mammalian expression systems can be used for transient or stable expression, with ultra high-yield protein expression. However, high yields are only possible in suspension cultures and more demanding culture conditions. Insect cultures are the same as mammalian, except that they can be used as both static and suspension cultures. These cultures also have demanding culture conditions and may also be time-consuming. Yeast cultures can produce eukaryotic proteins and are scalable, with minimum culture requirements. Yeast cultures may require growth culture optimization. Bacterial cultures are simple, scalable and low cost, but these may require protein-specific optimization and are not suitable for all mammalian proteins. Algal cultures are optimized for robust selection and expression, but these are less developed than other host platforms. Cell-free systems are open, free of any unnatural compounds, fast and simple. This system is, however, not optimal for scaling up.

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Found 3 matching solutions for this experiment

pMCSG7-D1aerobic

Ewa Niedzialkowska, Jerzy Haber Institute of Catalysis and Surfa

Upstream tips	Protocol tips	Downstream tips
	For expression under aerobic conditions BL21 (DE3)RILP cells harboring the pMCSG7 plasmid with the D1 gene were grown in 50 mL of Lennox LB medium (#LBL405, BioShop Canada Inc.) with 100 μg/mL ampicillin (#K029.4, Carl Roth) and 34 μg/mL chloramphenicol (#227920250, Acros Organics) overnight at 37 °C with shaking. The next day, the culture was transferred to 4 L of Terrific-Broth (TB) medium (#TER409, BioShop Canada Inc.) with ampicillin and chloramphenicol for selection, and cells were grown at 37 °C until the culture reached an OD600 of 1.2. The temperature was decreased to 18 °C, and cells were induced with 0.15 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) (#CN08.3, Carl Roth).

Protocol tips
For expression under aerobic conditions BL21 (DE3)RILP cells harboring the pMCSG7 plasmid with the D1 gene were grown in 50 mL of Lennox LB medium (#LBL405, BioShop Canada Inc.) with 100 μg/mL ampicillin (#K029.4, Carl Roth) and 34 μg/mL chloramphenicol (#227920250, Acros Organics) overnight at 37 °C with shaking. The next day, the culture was transferred to 4 L of Terrific-Broth (TB) medium (#TER409, BioShop Canada Inc.) with ampicillin and chloramphenicol for selection, and cells were grown at 37 °C until the culture reached an OD600 of 1.2. The temperature was decreased to 18 °C, and cells were induced with 0.15 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) (#CN08.3, Carl Roth).

Protocol tips

For expression under aerobic conditions BL21 (DE3)RILP cells harboring the pMCSG7 plasmid with the D1 gene were grown in 50 mL of Lennox LB medium (#LBL405, BioShop Canada Inc.) with 100 μg/mL ampicillin (#K029.4, Carl Roth) and 34 μg/mL chloramphenicol (#227920250, Acros Organics) overnight at 37 °C with shaking. The next day, the culture was transferred to 4 L of Terrific-Broth (TB) medium (#TER409, BioShop Canada Inc.) with ampicillin and chloramphenicol for selection, and cells were grown at 37 °C until the culture reached an OD600 of 1.2. The temperature was decreased to 18 °C, and cells were induced with 0.15 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) (#CN08.3, Carl Roth).

Publication protocol 1 Relevant paper

pASG-IBA5

Ewa Niedzialkowska, Jerzy Haber Institute of Catalysis and Surfa

Upstream tips	Protocol tips	Downstream tips
	For anaerobic cultures, the 400 mL of LB or M9ZB media were prepared in a 500 mL bottle that was closed with a rubber stopper, degassed with N2 and sterilized by autoclaving. The overnight aerobic culture was transferred to the medium through a syringe, without opening the bottle. In the case when the M9ZB medium was tested, cells were grown at 37 °C with shaking, and induced with AHT (0.2 μg/mL final) when the culture reached an OD600 of 0.8. When cells grew anaerobically in LB medium, they were induced at an OD600 of 0.5. The culture was supplemented with Na2MoO4 (10 mM final concentration) 15 min before induction with AHT (0.2 μg/mL final). The culture was grown for 2 h at 37 °C and then cells were pelleted. The pellet was stored at −80 °C for a few days before purification.

Protocol tips
For anaerobic cultures, the 400 mL of LB or M9ZB media were prepared in a 500 mL bottle that was closed with a rubber stopper, degassed with N2 and sterilized by autoclaving. The overnight aerobic culture was transferred to the medium through a syringe, without opening the bottle. In the case when the M9ZB medium was tested, cells were grown at 37 °C with shaking, and induced with AHT (0.2 μg/mL final) when the culture reached an OD600 of 0.8. When cells grew anaerobically in LB medium, they were induced at an OD600 of 0.5. The culture was supplemented with Na2MoO4 (10 mM final concentration) 15 min before induction with AHT (0.2 μg/mL final). The culture was grown for 2 h at 37 °C and then cells were pelleted. The pellet was stored at −80 °C for a few days before purification.

Protocol tips

For anaerobic cultures, the 400 mL of LB or M9ZB media were prepared in a 500 mL bottle that was closed with a rubber stopper, degassed with N2 and sterilized by autoclaving. The overnight aerobic culture was transferred to the medium through a syringe, without opening the bottle. In the case when the M9ZB medium was tested, cells were grown at 37 °C with shaking, and induced with AHT (0.2 μg/mL final) when the culture reached an OD600 of 0.8. When cells grew anaerobically in LB medium, they were induced at an OD600 of 0.5. The culture was supplemented with Na2MoO4 (10 mM final concentration) 15 min before induction with AHT (0.2 μg/mL final). The culture was grown for 2 h at 37 °C and then cells were pelleted. The pellet was stored at −80 °C for a few days before purification.

Publication protocol 1 Relevant paper

pASG-IBA35

Ewa Niedzialkowska, Jerzy Haber Institute of Catalysis and Surfa

Upstream tips	Protocol tips	Downstream tips
	For anaerobic cultures, the 400 mL of LB or M9ZB media were prepared in a 500 mL bottle that was closed with a rubber stopper, degassed with N2 and sterilized by autoclaving. The overnight aerobic culture was transferred to the medium through a syringe, without opening the bottle. In the case when the M9ZB medium was tested, cells were grown at 37 °C with shaking, and induced with AHT (0.2 μg/mL final) when the culture reached an OD600 of 0.8. When cells grew anaerobically in LB medium, they were induced at an OD600 of 0.5. The culture was supplemented with Na2MoO4 (10 mM final concentration) 15 min before induction with AHT (0.2 μg/mL final). The culture was grown for 2 h at 37 °C and then cells were pelleted. The pellet was stored at −80 °C for a few days before purification.

Protocol tips
For anaerobic cultures, the 400 mL of LB or M9ZB media were prepared in a 500 mL bottle that was closed with a rubber stopper, degassed with N2 and sterilized by autoclaving. The overnight aerobic culture was transferred to the medium through a syringe, without opening the bottle. In the case when the M9ZB medium was tested, cells were grown at 37 °C with shaking, and induced with AHT (0.2 μg/mL final) when the culture reached an OD600 of 0.8. When cells grew anaerobically in LB medium, they were induced at an OD600 of 0.5. The culture was supplemented with Na2MoO4 (10 mM final concentration) 15 min before induction with AHT (0.2 μg/mL final). The culture was grown for 2 h at 37 °C and then cells were pelleted. The pellet was stored at −80 °C for a few days before purification.

Protocol tips

Publication protocol 1 Relevant paper

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