In general, T7 is stronger in producing mRNA. However, the final amount of protein produced depends on other factors like protein solubility, stability, and toxicity.
Here, https://microbialcellfactories.biomedcentral.com/articles/10.1186/1475-2859-12-26, is a study that compares T7 and other promoters for the production of few different proteins.
In general, T7 is stronger in producing mRNA. However, the final amount of protein produced depends on other factors like protein solubility, stability, and toxicity.Here, https://microbialcellfactories.biomedcentral.com/articles/10.1186/1475-2859-12-26, is a study that compares T7 and other promoters for the production of few different proteins.
The best reference I could find is a book chapter (available here as a .pdf) from a book on optimizing the expression of membrane proteins that argues for T7. Although they don't appear to reference a source for that specific claim, some of the references close by could be helpful. It seems as though this applies in general, not just to membrane proteins. They bring up limitations of the araBAD system as well:
However, careful studies of the araBAD promoter by Siegele et al. [20] and Giacalone et al. [21] both agreed that at subsaturating levels of l- arabinose, protein expression cultures contain a mixed population with only some of the cells expressing protein. In addition, the potential for protein overexpression is generally lower when a pBAD vector is compared to T7-mediated expression from a pET construct.
The best reference I could find is a book chapter (available here as a .pdf) from a book on optimizing the expression of membrane proteins that argues for T7. Although they don't appear to reference a source for that specific claim, some of the references close by could be helpful. It seems as though this applies in general, not just to membrane proteins. They bring up limitations of the araBAD system as well:
However, careful studies of the araBAD promoter by Siegele et al. [20] and Giacalone et al. [21] both agreed that at subsaturating levels of l- arabinose, protein expression cultures contain a mixed population with only some of the cells expressing protein. In addition, the potential for protein overexpression is generally lower when a pBAD vector is compared to T7-mediated expression from a pET construct.
The exact numbers depend on the protein.
In my hands, I would say between the pAraBAD and a pLac-T5 you can expect some fold improvement and pAraBAD and a pT7 promoters you can expect about an order of magnitude… but there are two big caveats.
First, pET and other T7 driven plasmids do not work in K-12 strains (without the pTARA) so BL21 is generally used. BL21 is much better generally than K-12 strains (more primary metabolites cf.omics paper) for a lot of things including protein expression. The chaperone balance is difference, so you will get differences from that too —if you have used the TAKARA plasmid team you will have notice how quirky protein expression optimisation can be.
Second, if your protein has a cofactor you have a major problem. FeS clusters, PLP, heme or even O2 (for maturation of GFP, say) become rate limiting and overexpressing the protein can make it highly toxic and you might be in a bigger pickle than with a lower level promoter. In my thesis, I added pyridoxine to a pBAD expression, because it gave an improvement.
Footnote: If the expression levels are really bad, changing promoter might not be that great as the assumption of linearity might not hold.
If heterologous and not codon optimised, consider using pRARE.
If insoluble, consider using ethanol shock and the TAKARA chaperon plasmid team.
If oxygen sensitive, alas, doing expression and purification anaerobically.
The exact numbers depend on the protein. In my hands, I would say between the pAraBAD and a pLac-T5 you can expect some fold improvement and pAraBAD and a pT7 promoters you can expect about an order of magnitude… but there are two big caveats. First, pET and other T7 driven plasmids do not work in K-12 strains (without the pTARA) so BL21 is generally used. BL21 is much better generally than K-12 strains (more primary metabolites cf.omics paper) for a lot of things including protein expression. The chaperone balance is difference, so you will get differences from that too —if you have used the TAKARA plasmid team you will have notice how quirky protein expression optimisation can be. Second, if your protein has a cofactor you have a major problem. FeS clusters, PLP, heme or even O2 (for maturation of GFP, say) become rate limiting and overexpressing the protein can make it highly toxic and you might be in a bigger pickle than with a lower level promoter. In my thesis, I added pyridoxine to a pBAD expression, because it gave an improvement.
Footnote: If the expression levels are really bad, changing promoter might not be that great as the assumption of linearity might not hold. If heterologous and not codon optimised, consider using pRARE. If insoluble, consider using ethanol shock and the TAKARA chaperon plasmid team. If oxygen sensitive, alas, doing expression and purification anaerobically.
thanks for taking a stab at this old question... I know that it may be difficult, but do you think you could add some links/references to some of the details you mention in your answer? We always appreciate references/links on BiologySE - and it will definitely improve the strength of your answer. Thanks!More
In general, T7 is stronger in producing mRNA. However, the final amount of protein produced depends on other factors like protein solubility, stability, and toxicity. Here, https://microbialcellfactories.biomedcentral.com/articles/10.1186/1475-2859-12-26, is a study that compares T7 and other promoters for the production of few different proteins.
In general, T7 is stronger in producing mRNA. However, the final amount of protein produced depends on other factors like protein solubility, stability, and toxicity.Here, https://microbialcellfactories.biomedcentral.com/articles/10.1186/1475-2859-12-26, is a study that compares T7 and other promoters for the production of few different proteins.
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The best reference I could find is a book chapter (available here as a .pdf) from a book on optimizing the expression of membrane proteins that argues for T7. Although they don't appear to reference a source for that specific claim, some of the references close by could be helpful. It seems as though this applies in general, not just to membrane proteins. They bring up limitations of the araBAD system as well:
The best reference I could find is a book chapter (available here as a .pdf) from a book on optimizing the expression of membrane proteins that argues for T7. Although they don't appear to reference a source for that specific claim, some of the references close by could be helpful. It seems as though this applies in general, not just to membrane proteins. They bring up limitations of the araBAD system as well:
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The exact numbers depend on the protein.
In my hands, I would say between the pAraBAD and a pLac-T5 you can expect some fold improvement and pAraBAD and a pT7 promoters you can expect about an order of magnitude… but there are two big caveats.
First, pET and other T7 driven plasmids do not work in K-12 strains (without the pTARA) so BL21 is generally used. BL21 is much better generally than K-12 strains (more primary metabolites cf. omics paper) for a lot of things including protein expression. The chaperone balance is difference, so you will get differences from that too —if you have used the TAKARA plasmid team you will have notice how quirky protein expression optimisation can be.
Second, if your protein has a cofactor you have a major problem. FeS clusters, PLP, heme or even O2 (for maturation of GFP, say) become rate limiting and overexpressing the protein can make it highly toxic and you might be in a bigger pickle than with a lower level promoter. In my thesis, I added pyridoxine to a pBAD expression, because it gave an improvement.
Footnote: If the expression levels are really bad, changing promoter might not be that great as the assumption of linearity might not hold.
If heterologous and not codon optimised, consider using pRARE.
If insoluble, consider using ethanol shock and the TAKARA chaperon plasmid team.
If oxygen sensitive, alas, doing expression and purification anaerobically.
The exact numbers depend on the protein.
In my hands, I would say between the pAraBAD and a pLac-T5 you can expect some fold improvement and pAraBAD and a pT7 promoters you can expect about an order of magnitude… but there are two big caveats.
First, pET and other T7 driven plasmids do not work in K-12 strains (without the pTARA) so BL21 is generally used. BL21 is much better generally than K-12 strains (more primary metabolites cf. omics paper) for a lot of things including protein expression. The chaperone balance is difference, so you will get differences from that too —if you have used the TAKARA plasmid team you will have notice how quirky protein expression optimisation can be.
Second, if your protein has a cofactor you have a major problem. FeS clusters, PLP, heme or even O2 (for maturation of GFP, say) become rate limiting and overexpressing the protein can make it highly toxic and you might be in a bigger pickle than with a lower level promoter. In my thesis, I added pyridoxine to a pBAD expression, because it gave an improvement.
Footnote: If the expression levels are really bad, changing promoter might not be that great as the assumption of linearity might not hold.
If heterologous and not codon optimised, consider using pRARE.
If insoluble, consider using ethanol shock and the TAKARA chaperon plasmid team.
If oxygen sensitive, alas, doing expression and purification anaerobically.
More
VOTE
VOTE