The protein that I am working on has a tendency to form inclusion bodies. So, I want to know that there is anything that we can add or any precaution that we can take during protein expression that will further reduce the chances of the protein to be expressed as inclusion bodies.
OR
Following the refolding protocol the only solution to this problem?
Hi!! Deekshi,
You can try these methods to increase the solubility level of your protein:
(1). You can use 10%-20% of glycerol in your media. Viscosity provided by glycerol lowers the growth and helps in solubility.
(2). Try to induce your recombinant protein with low concentration of IPTG such as 0.1-0.5 mM IPTG.
(3). I have faced similar situation several times there i have changed growth media, sometimes it works. try to optimize the solubility in different media such as LB, 2XYT, Terrific Growth, S9 etc. You need to optimize the different concentration of IPTG for different media.
(4). One of my protein i got soluble in AIM (Auto induction Media). Its quite good for high yield soluble protein. There are different AIM media available such as AIM-LB, AIM,2XYT, AIM-TB. Try these, i am sure you will get good response.
(5). Lower down the temp to 16-23 degree after induction, Sometimes lowering the temp works better.
(6). You can use 1-3% of ethanol, In some cases ethanol is reported to decrease the inclusion bodies formation. Add 1-3% of ethanol while inoculating the culture. Growth will be less which ultimately helps in solubility.
(7).In some cases less amount or less concentration of osmolytes such as glucose, sorbitol, mannitol etc can helps in solubilty. Start with less concentration of these osmolytes.
(8). You can also try the heat shock method to improve your solubility. Grow your culture till 0.2 OD in 37 degree then grow it at 42 degree till 0.6 OD then divide it into 3 fraction induced with 0.5 mM IPTG and incubate at 23 degree and 37 degree 42 degree for overnight, 5 hrs and 3 hrs respectively.
(9). Try different expression host according to your promoter for T-5 use M-15, SG13009, XL-GOLD, XL-BLUE, TG-1 for T-7 use BL-21, BL21(DE3), ROSETTA, CODON PLUS etc. there are various cells. optimization depends upon the availability of these cells at your lab.
(10). Optimize all condition if you won't get any positive response then ultimately you need to change the Tag or vector.
I hope anyone of above methods should click for your work.
For any further queries you are welcome.
Good Luck.
Like Rajeev said try lower temperatures or lower IPTG concentration. Try also different expression strains. I suggest doing huge expression experiment where you would test different strains, temperatures, IPTG concentrations and time of induction. It might help.
Hi!! Deekshi,
You can try these methods to increase the solubility level of your protein:
(1). You can use 10%-20% of glycerol in your media. Viscosity provided by glycerol lowers the growth and helps in solubility.
(2). Try to induce your recombinant protein with low concentration of IPTG such as 0.1-0.5 mM IPTG.
(3). I have faced similar situation several times there i have changed growth media, sometimes it works. try to optimize the solubility in different media such as LB, 2XYT, Terrific Growth, S9 etc. You need to optimize the different concentration of IPTG for different media.
(4). One of my protein i got soluble in AIM (Auto induction Media). Its quite good for high yield soluble protein. There are different AIM media available such as AIM-LB, AIM,2XYT, AIM-TB. Try these, i am sure you will get good response.
(5). Lower down the temp to 16-23 degree after induction, Sometimes lowering the temp works better.
(6). You can use 1-3% of ethanol, In some cases ethanol is reported to decrease the inclusion bodies formation. Add 1-3% of ethanol while inoculating the culture. Growth will be less which ultimately helps in solubility.
(7).In some cases less amount or less concentration of osmolytes such as glucose, sorbitol, mannitol etc can helps in solubilty. Start with less concentration of these osmolytes.
(8). You can also try the heat shock method to improve your solubility. Grow your culture till 0.2 OD in 37 degree then grow it at 42 degree till 0.6 OD then divide it into 3 fraction induced with 0.5 mM IPTG and incubate at 23 degree and 37 degree 42 degree for overnight, 5 hrs and 3 hrs respectively.
(9). Try different expression host according to your promoter for T-5 use M-15, SG13009, XL-GOLD, XL-BLUE, TG-1 for T-7 use BL-21, BL21(DE3), ROSETTA, CODON PLUS etc. there are various cells. optimization depends upon the availability of these cells at your lab.
(10). Optimize all condition if you won't get any positive response then ultimately you need to change the Tag or vector.
I hope anyone of above methods should click for your work.
For any further queries you are welcome.
Good Luck.
If manipulating expression conditions (temp, inducer conc, etc as described above) doesn't help, you could try chaperone co-expression. The Takara plasmid set (from Clontech I think) is a good place to start if it's compatible with your expressions system.
Try to get expression at high temperature i.e. it will produce Chapronen proteins in the organism, may help in proper folding of your proteins. Moreover you can dissolve recombinant proteins in urea and then fractional diffusion, you can eliminate urea from your buffer. You may use DTT for this purpose.
Decreasing the temperature during expression phase is generally a good solution (and the easiest :)) i.e. increasing biomass first to an OD600 nm of about 1 or 2 by growing cells at 37°C and add IPTG after decreasing the temperature of the culture at about 16-18°C (you could also try lower temperature but you have to follow the expression level over time). Note that expression in E.coli with classical pET system (T7 RNA pol) are very efficient so you have to be sure that your culture is really at 16-18°C before adding the IPTG. If you are doing large volumes of culture, you may need some minutes to be at the right temperature.
IB formation is often a result of a too large amount of overexpressed protein. As T7 RNA Pol is very (too !!) efficient, sometimes, it has the tendency to overflow the bacteria expression/folding system. Try to use different promoters (you have to dive again in molecular biology) like constitutives promoters or inducible promoters not depending on the T7 RNA Pol to decrease the expression level.
Addition of some additives to the medium (chemical chaperonne) could be also a solution. Famous additives are glycerol, sorbitol or bétaïne.
I am not convinced by the possibility to regulate expression by manipulating the concentration of IPTG. In our hands here, going from 0.1 to 2 mM has no effect on the expression level especially with pET systems.
try expressing @5~10C using the arctic express competent cells. and using low concentration of IPTG.
just google "arctic express competent cells protocol"
or you can try auto-induction
If your protein refolds, then refolding protocol is the best way to maximize your yield. You can minimize inclusion body formation but you can not completely prevent it.
Moreover, the refolding protocol results in high purity protein. In our lab we make His6-tagged proteins and refold them on the Ni-column.
All the best!
Everyone in this discussion has suggested excellent solutions. you have to check what is fit in your case. I have one counter argument. inclusion bodies are mostly made up only one protein. infact isolating IBs could be an easier way to isolate your desired protein. I will suggest you to read papers of DR Amulya panda, NII, New Delhi. See whether it can work for your protein also.
Thanks everyone for your valuable suggestions. I will surely consider them.
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