I am working with tail tubular proteins from Y. enterocolitica-specific phages. These are small proteins, approximately ~20 kDa, they are fused with MBP, which gives 60 kDa. Transformation went successful, I am sure that recombinants do have construct inside them. I induced at OD600 = 0.6 with 0.1 mM IPTG, incubated it overnight at 12C after induction. SDS-PAGE didn't show expression not only in both proteins, but also not in the supernatant, not in the pellet. I noticed that OD600 lowered after induction, so I decided to lower the concentration of IPTG; I used 0.05 mM. Hardly did it help - OD600 didn't decrease, but stopped at 0.6. I also tried the 1-day procedure, in which I induced cells with the same concentration of IPTG, but at the temperature of 37C for 3 hours. It didn't help as well, there was no expression. My friend who works with tail tubular protein from Klebsiella-specific phage has the same problem.
Hi Natalia,
Is that possible the phage protein is toxic to the BL21 (DE3)? The leaky effect of BL21 (DE3) maybe produce slight target protein during bacterial replication and interfere their growth. To avoid the leaky effect of BL21 (DE3), try the BL21 (DE3)pLysS competent cell.
Good luck
Please check the frame of the insert in the recombinant vector. if the gene is in Frame and you facing same problem than change the bacterial strains that are specialized for expression of toxic genes (you can get them in NEB and thermofisher).
hope you will see the desired. good luck
Hello Natalia,
do you have any particular reason why do you express the protein at 12ºC and only with 0.1mM IPTG concentration?
Usual IPTG concentration ranges from 0.1 to 1 mM, so maybe you can use 0.5mM. Next, I am not sure if BL21(DE3) strain is happy with as low temperature as 12 ºC. I would go for 18 ºC at the minimum and test the expression for 12, 24, and even 36 hours. On the other hand, there is an E. coli ArcticExpress strain engineered for low-temperature expression - they are fine with 12 ºC. ArcticExpress also co-expresses chaperonins to aid protein folding, in case the protein of interest has some solubility issues.
You should be also aware that 3/4 of all the supplements will be used to produce 60kDa MBP, and only 1/4 will be left to build your 20kDa phage protein. Therefore, if the protein yield won't be sufficient even after optimisation of expression, you may consider switching to smaller fusion partner, e.g. thioredoxin or SUMO.
I hope I helped a little :)
Best regards,
Jan
Thank you for all the advice! I forgot to add that I also tried using pLysS cells. I had the same problem: there was no expression, OD600 lowered after induction or cells just stopped growing. These conditions (12C overnight, 0.1 mM IPTG) were suggested by my supervisor. She used them to express other tail tubular proteins (she had different constructs and different vector). Also, it should be noted that I used ligation-independent cloning (LIC) system to create a construct.
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