I have been trying to express some large eukaryotic proteins,over 100kDa in sizes, in BL21(DE3) and Rosetta(DE3), the outcome is frustrating.Does anybody have any ideas about how to achieve this goal?
Dear Lin,
based on my experiences as a research scientists and later as a R&D Manager in a company specialized to high cell density growth systems for recombinant protein expression, I would like to point out some generic facts causing the difficulties with large proteins:
1) The process of recombinant protein production in E. coli is very fast, especially with the T7-based polymerase system. Some other expression systems may possibly give better results. Roughly said, protein is produced 5 times faster in E. coli compared to eukaryotic organisms.
2) fast protein synthesis causes problems in protein folding, since the folding machinery simply cannot match the translational capacity. You could try the co-expression of different chaperones
3) Protein synthesis rate is related to growth rate. Accordingly, any method which can slow down the protein synthesis rate may help. Lowering the expression temperature is a common approach. If you have the possibility to use bioreactors (very few of us has ), you can apply fed-batch technology to control the growth rate.
4) the use of a minimal amount of inducer (IPTG) may help, sometimes the best results can be obtained by omitting completely IPTG and putting the trust on expression leakage
5) Selection of a proper medium may play a big role. Some proteins express nicely in LB but not in TB. Expression in mineral salt medium composition like M9 may give surprisingly good results, although the cell yields are usually low.
6) Last, I would suggest considering BioSilta's growth systems which facilitate high cell densities, long expression phases (24 h instead of 3 h) and control of growth and protein synthesis. I have realized that many researcher who are disappointed with the recombinant protein yields, tend to go for codon optimization, which actually will accelerate the rate of protein synthesis instead of decelerating it. Some constructs will never express in E. coli, but some may express well in alternative medium solutions. Regarding the amount of work needed for reconstructing new clones is much higher than the work of testing different cultivation media in deepwell plates or even in shake flasks.
With kind regards, Antti Vasala
Hi! what do you mean with frustrating? low expression, degradation, misfolding or no expression at all? do you think that those protein are toxic?
if you could, you should try with Pichia pastoris, which is very easy to handle and inexpensive as E. coli and it is more likely to express high molecular weight proteins.
In E. coli you could try with pLys strains or Arctic E. coli, which express chaperonins!
@Valentina Piano,frustrating means no expression.I have also tried C41(DE3) ,a strain for expression toxic proteins,and no expression again,
@Surendra Vikram,I have tried different concentration of IPTG range from 0.1mM to 1mM;different inducing time span from 3 to 12 hours ,and I also tried to express them in 25 ℃,and no expression were achieved.
@Surendra Vikram
Can I use pET28a plasmid directly in cell free expression system?
just out of curiosity: what kind of protein are you trying to express? (and I am curious because I am expressing protein of similar size in eukaryots and those also are not the happiest cells under the sun with such burden). :)
Dear Lin,
based on my experiences as a research scientists and later as a R&D Manager in a company specialized to high cell density growth systems for recombinant protein expression, I would like to point out some generic facts causing the difficulties with large proteins:
1) The process of recombinant protein production in E. coli is very fast, especially with the T7-based polymerase system. Some other expression systems may possibly give better results. Roughly said, protein is produced 5 times faster in E. coli compared to eukaryotic organisms.
2) fast protein synthesis causes problems in protein folding, since the folding machinery simply cannot match the translational capacity. You could try the co-expression of different chaperones
3) Protein synthesis rate is related to growth rate. Accordingly, any method which can slow down the protein synthesis rate may help. Lowering the expression temperature is a common approach. If you have the possibility to use bioreactors (very few of us has ), you can apply fed-batch technology to control the growth rate.
4) the use of a minimal amount of inducer (IPTG) may help, sometimes the best results can be obtained by omitting completely IPTG and putting the trust on expression leakage
5) Selection of a proper medium may play a big role. Some proteins express nicely in LB but not in TB. Expression in mineral salt medium composition like M9 may give surprisingly good results, although the cell yields are usually low.
6) Last, I would suggest considering BioSilta's growth systems which facilitate high cell densities, long expression phases (24 h instead of 3 h) and control of growth and protein synthesis. I have realized that many researcher who are disappointed with the recombinant protein yields, tend to go for codon optimization, which actually will accelerate the rate of protein synthesis instead of decelerating it. Some constructs will never express in E. coli, but some may express well in alternative medium solutions. Regarding the amount of work needed for reconstructing new clones is much higher than the work of testing different cultivation media in deepwell plates or even in shake flasks.
With kind regards, Antti Vasala
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