I am working with leishmania threonine synthase (LdTS) enzyme of aspartic acid pathway which is ~75kDa protein. I purified the protein and confirmed by western blot too. The condition used for induction is -
0.1mM IPTG concentration at 20°C for 12hours.
For purification I am using Tris buffer and PBS buffer both with Imidazole concentration as by Ni2+ NTA chromatography-
Wash I 5mM
Wash II 10mM
Elution 250mM
The problem which I am facing is that I am non specific bands along with my band of interest. How get rid of this? As well as how I can improve my yield? Currently my yield is 4.146mg/L with Tris and 3.886mg/L with PBS buffer.
I have attached the gel images and blot image below.
Dear Zulfa Nasir
just some preliminary questions:
1) why did you use so low temperature and iptg amount? at higher temperature and IPTG amounts you protein was overexpressed but in the non soluble form?
2) At which O.D(600nm) and in which media did you performed the induction?
3) Did you clone the gene directly from the leishmania genome or did you cloned a sintetic codon optimized DNA fragment?
Because i think that you can try different options
1) Use an high cell density expression media, which will allow to not improve the specific expression but the volumetric ones.
you can find some example of this on the following link of my blog:
https://www.blogger.com/video.g?token=AD6v5dwU8Sl-43BfVTSeIRFBcQiWXAeg80Ud5UCxiXqUj4AFUO7p1BOHHmV4NiV0MY32A41mTgUH2-nyouDVBhcVFOuGc1lJ5_1WgP3RrsvakB6iJHCW_JOnrD8kIeiUSimWYahOs-OP
https://www.blogger.com/video.g?token=AD6v5dzLWqhoYwVoIC07Aj1G4RRYNxwM3Ndyk09KE3rmvGIdgfhR6WOBAH6TE0sDThwNquI9hZ9u7vKUDdMsusqQ3IcouXA53Y37DqhPmlYX4e9_Jnz5YS-87K68gQcCTjBMvNE4MlI
2)Try to reduce the codon bias by produce a new clone using a codon optimized DNA fragment or try to use different e.coli strains (e.g Rosetta or BL21 codon +)
3)Add an N-terminal fusion tag (eg TRx, GB1) to your construct to pump the expression level.
4) Did you used standard pet vectors? Maybe you can try the takara pcold, which work well at so low induction temperature.
you can find some example of how the addition of fusion tag on the following link
https://www.blogger.com/video.g?token=AD6v5dzPeA9ws3hcPvrO8fBABN8iFQ0dwI7GIHys9p8dwVtov4dGrKXH9PF91d9bVMYcLxTYY3XJesKt62Y-LsZcaHDCJKZbNW-DLUm_4p5-75Dbj1VZlKTlyyl2aQzf-EPwO3Ac6eBG
but to hypotize which could be the most promising one we need to know more about the trials that you perfromed to set up the current protocol.
best
Manuele
Most people have addressed all queries. I suggest you modify your work as per their suggestion. I would just similar suggestion-
Modify media composition to reduce the amount of nonspecific proteins
and
Optimize the concentration of IPTG (refer literature review)
You can increase the purity by increasing length of the 6XHis tag allowing for stronger imidazole wash and greater concentration of imidazole to elute your polyhistidine tagged protein . How many Histidines do you want? You can add up to an additional 6 histidines (If you already have 6 then you can have 12. If you want another 3 you can have 9) using the following method:
1. Design Forward and Reverse Primers Inverse Primers that amplify the plasmid backbone facing away from each other. The primers contain a gap and overlap distance of zero bases for no frameshift errors.
The forward primer contains near the 5'-overhang or in the middle at (CAC)x6 insertion to add 6 histidines. The reverse primer contains no mutations.
Phosphorylate the primers with T4 polynucleotide kinase + ATP + Mg2+
Perform the PCR with the phosphorylated primers on your wildtype plasmid DNA. See if you get PCR product of the size of your plasmid on a gel. If so then DPNI digestion and then perform gel purification or PCR product purification.
Blunt end ligate the phosphorylated PCR product together with T4 DNA ligase + ATP + Mg2+.
Transform the ligation reaction to E.coli cells, plate on appropriate antibiotic agar plates, select colonies to purify the plasmids from and DNA sequence the plasmids from those colonies.
If the PCR worked and the ligation worked then one of the colonies will have the intended mutagenic insertion of however many extra histidines you wanted. Just like with site directed mutagenesis it is still possible to get huge primer insertions so make sure you sequence the clones and select the right clones.
I have done this so I know it works.
Honestly, your yield is pretty good, just scale it up. It takes about the same amount of time to do a 6 liter induction vs a 1 L induction.
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