Protein Expression Prokaryotic cells - E. coli PduP-E bacteriophage ϕX174

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

pMCY85

Mimi C. Yung, Biosciences and Biotechnology Division, Physical a

Upstream tips	Protocol tips	Downstream tips
For E expression experiments, E. coli cells were pre-cultured from single colonies in 5 mL of LB supplemented with appropriate antibiotic(s) at 25 °C for 16 h. Cells were then diluted into 10 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 50-mL flask and were cultured at 30 °C and 220 rpm. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to the cultures to a final concentration of 0.5 mM at the start of growth (t = −3 h) or once the cultures reached an OD600 of ~0.4 (t = 0) where indicated. Rhamnose was added to the cultures to final concentrations of 0.1, 0.2, or 0.5 mM once the cultures reached an OD600 of ~0.4.	For isolation of BMCs and purification of PduP-E, E. coli cells were pre-cultured from single colonies in 20 mL of LB supplemented with appropriate antibiotic(s) in a 250-mL flask at 25 °C for 16 h. Cells were then diluted into 200 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 1-L flask. Cells were cultured at 30 °C and 220 rpm and harvested at 3 h post-rhamnose induction. All induction conditions with IPTG and rhamnose are the same as above.

Upstream tips
For E expression experiments, E. coli cells were pre-cultured from single colonies in 5 mL of LB supplemented with appropriate antibiotic(s) at 25 °C for 16 h. Cells were then diluted into 10 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 50-mL flask and were cultured at 30 °C and 220 rpm. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to the cultures to a final concentration of 0.5 mM at the start of growth (t = −3 h) or once the cultures reached an OD600 of ~0.4 (t = 0) where indicated. Rhamnose was added to the cultures to final concentrations of 0.1, 0.2, or 0.5 mM once the cultures reached an OD600 of ~0.4.
Protocol tips
For isolation of BMCs and purification of PduP-E, E. coli cells were pre-cultured from single colonies in 20 mL of LB supplemented with appropriate antibiotic(s) in a 250-mL flask at 25 °C for 16 h. Cells were then diluted into 200 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 1-L flask. Cells were cultured at 30 °C and 220 rpm and harvested at 3 h post-rhamnose induction. All induction conditions with IPTG and rhamnose are the same as above.

Publication protocol 1 Relevant paper

pMCY86

Mimi C. Yung, Biosciences and Biotechnology Division, Physical a

Upstream tips	Protocol tips	Downstream tips
For E expression experiments, E. coli cells were pre-cultured from single colonies in 5 mL of LB supplemented with appropriate antibiotic(s) at 25 °C for 16 h. Cells were then diluted into 10 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 50-mL flask and were cultured at 30 °C and 220 rpm. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to the cultures to a final concentration of 0.5 mM at the start of growth (t = −3 h) or once the cultures reached an OD600 of ~0.4 (t = 0) where indicated. Rhamnose was added to the cultures to final concentrations of 0.1, 0.2, or 0.5 mM once the cultures reached an OD600 of ~0.4.	For isolation of BMCs and purification of PduP-E, E. coli cells were pre-cultured from single colonies in 20 mL of LB supplemented with appropriate antibiotic(s) in a 250-mL flask at 25 °C for 16 h. Cells were then diluted into 200 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 1-L flask. Cells were cultured at 30 °C and 220 rpm and harvested at 3 h post-rhamnose induction. All induction conditions with IPTG and rhamnose are the same as above.

Upstream tips
For E expression experiments, E. coli cells were pre-cultured from single colonies in 5 mL of LB supplemented with appropriate antibiotic(s) at 25 °C for 16 h. Cells were then diluted into 10 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 50-mL flask and were cultured at 30 °C and 220 rpm. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to the cultures to a final concentration of 0.5 mM at the start of growth (t = −3 h) or once the cultures reached an OD600 of ~0.4 (t = 0) where indicated. Rhamnose was added to the cultures to final concentrations of 0.1, 0.2, or 0.5 mM once the cultures reached an OD600 of ~0.4.
Protocol tips
For isolation of BMCs and purification of PduP-E, E. coli cells were pre-cultured from single colonies in 20 mL of LB supplemented with appropriate antibiotic(s) in a 250-mL flask at 25 °C for 16 h. Cells were then diluted into 200 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 1-L flask. Cells were cultured at 30 °C and 220 rpm and harvested at 3 h post-rhamnose induction. All induction conditions with IPTG and rhamnose are the same as above.

Publication protocol 1 Relevant paper

pMCY87

Mimi C. Yung, Biosciences and Biotechnology Division, Physical a

Upstream tips	Protocol tips	Downstream tips
For E expression experiments, E. coli cells were pre-cultured from single colonies in 5 mL of LB supplemented with appropriate antibiotic(s) at 25 °C for 16 h. Cells were then diluted into 10 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 50-mL flask and were cultured at 30 °C and 220 rpm. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to the cultures to a final concentration of 0.5 mM at the start of growth (t = −3 h) or once the cultures reached an OD600 of ~0.4 (t = 0) where indicated. Rhamnose was added to the cultures to final concentrations of 0.1, 0.2, or 0.5 mM once the cultures reached an OD600 of ~0.4.	For isolation of BMCs and purification of PduP-E, E. coli cells were pre-cultured from single colonies in 20 mL of LB supplemented with appropriate antibiotic(s) in a 250-mL flask at 25 °C for 16 h. Cells were then diluted into 200 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 1-L flask. Cells were cultured at 30 °C and 220 rpm and harvested at 3 h post-rhamnose induction. All induction conditions with IPTG and rhamnose are the same as above.

Upstream tips
For E expression experiments, E. coli cells were pre-cultured from single colonies in 5 mL of LB supplemented with appropriate antibiotic(s) at 25 °C for 16 h. Cells were then diluted into 10 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 50-mL flask and were cultured at 30 °C and 220 rpm. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to the cultures to a final concentration of 0.5 mM at the start of growth (t = −3 h) or once the cultures reached an OD600 of ~0.4 (t = 0) where indicated. Rhamnose was added to the cultures to final concentrations of 0.1, 0.2, or 0.5 mM once the cultures reached an OD600 of ~0.4.
Protocol tips
For isolation of BMCs and purification of PduP-E, E. coli cells were pre-cultured from single colonies in 20 mL of LB supplemented with appropriate antibiotic(s) in a 250-mL flask at 25 °C for 16 h. Cells were then diluted into 200 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 1-L flask. Cells were cultured at 30 °C and 220 rpm and harvested at 3 h post-rhamnose induction. All induction conditions with IPTG and rhamnose are the same as above.

Publication protocol 1 Relevant paper

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