Hello esteemed scientists,
I am endeavoring to increase the yield of my recombinant protein. It is a 120 kDa protein tagged with MBP and features three intrinsically disordered regions with pI around 6.4. I have attempted expression in three different hosts—Rosetta D3M, BL21 DE3, and ArcticExpress—but the yield remains persistently low. Despite successful expression in all three hosts, the yield is less than 0.2 percent. Upon conducting Western blotting followed by detection with anti-MBP antibodies, I observed several bands with lower molecular weights alongside my anticipated band, suggesting possible degradation. Despite using a bacterial protease inhibitor cocktail alongside EDTA and PMSF, the root cause of this low yield remains elusive. It's worth noting that there was no protein of interest in the insoluble phase, indicating that protein solubility is not a problem. Additionally, in the case of misfolding, as our hosts express two chaperones which enhance protein folding, the issue might not be related to misfolding of our protein.
I have explored various IPTG concentrations ranging from 0.1 to 1 mM and post-induction incubation times ranging from 1 hour to 48 hours, alongside experimenting with different OD600 values at the time of induction. Despite these efforts, my yield remains unsatisfactory.
My overall process is as follows:
1-Double selection of transformed colonies with appropriate antibiotics followed by the outgrowth of 2.5 ml of overnight cultured bacteria to 250 ml LB medium containing corresponding antibiotics.
2-Expression induction by IPTG 0.3 mM at OD 0.7 while keeping the flask on ice, followed by continuation of the culture at 25 degrees Celsius and 250 rpm shaking.
3-Centrifugation at 4 degrees Celsius, 6000g for 15 minutes followed by flash freezing the pellets in liquid nitrogen.
4-Thawing the frozen pellets at room temperature and adding a lysis buffer containing 20 mM Tris, 0.2 mM NaCl, 1 mM EDTA, 1 mM DTT, 10 mM MgCl2, 0.2 mM PMSF, a protease inhibitor cocktail, and 1% Triton X100, with pH adjusted to 7.2 as the pI of my protein is 6.4.
5-Sonication on ice with 50% power for 15 seconds followed by a 30-second rest and centrifugation at 16,000g, 4 degrees Celsius for 15 minutes followed by overnight dialysis of the supernatant at a cold room.
6-Purification by an MBP purification column and elution with 10 mM maltose, then storing the protein solution at 4 degrees Celsius for no more than three weeks.
I would be immensely grateful if you could suggest ways to increase the overall yield or identify any potential issues with my protein or my protocol. Thank you.
Hi!
Your protocol is generally reasonable. What made me successful with recombinant expression in BL21 was amplifying the gene without the signal peptide.
Article Recombinant expression, purification, and characterization o...
@fabiane Cristina dos Santos
Thank you, Fabiane. This could definitely be helpful. Since my protein has three intrinsically disordered regions and is a cytoplasmic protein, so I am concerned about potential misfolding if the signal peptide is removed. Nonetheless, it's worth a try.
@Sirvan Abbasbeigi
Thank you, Sirvan, for your comprehensive answer. Actually, we have tried most of your advice before, but the yield still remains low. Right now, we are considering eliminating Triton X100 from our lysis buffer to omit the dialysis step and minimize the time period between cell lysis and purification as you mentioned. I hope this trick can work.
Hi Seyedsaber Mirabdali ,
what worked for me in the past, was addition of sorbitol and glycine betaine
onlinelibrary.wiley.com/doi/10.1016/0014-5793(91)81372-F/abstract
Also the recommended removal of signal peptide may be useful, but if your protein is cytosolic, it probably doesn't contain any.
Additionally, did you consider to express it with the tag on the other side or changing the tag or expression without tag altogether? Because the tag may clash with some part of the protein and cause trouble.
Alternatively, you may consider switching the expression host, e.g. to Pichia pastoris.
Tomáš Hluska
Hi Tomáš Hluska, Thank you for your expert recommendation. We previously expressed this protein with a GST tag, and the results were consistent. We tested both N-terminus and C-terminus tags, yielding similar results. Additionally, we are considering the addition of sorbitol, trehalose, or glycine betaine in the near future, as you suggested. It seems we may also need to explore alternative hosts.
Our lysis buffer includes Triton, necessitating overnight dialysis to mitigate its detrimental effect on the purification column. Do you have any insights on modifying the lysis buffer composition to eliminate the need for dialysis and shorten the purification process?
Seyedsaber Mirabdali you wrote that your protein is cytosolic, right? In that case you don't need any detergent. You only extract more proteins than neccessary (i.e. then you need to purify your protein more).
If you're not sure, whether the problem is with long dialysis, you can analyse on SDS-PAGE/Western blot directly your bacteria or the crude extract before dialysis. So you will see, whether there is much more of your protein.
Tomáš Hluska Actually, since my protein is highly prone to aggregation, we decided to use Triton to decrease the probability of aggregation. We analyzed the pre-dialysis and post-dialysis samples on SDS-PAGE, but they did not show much difference.
Seyedsaber Mirabdali if it's neccessary only to prevent aggregation, you may use lower concentration than what is used for membrane dissolution. Or try different detergent.
However, it seems that Triton and dialysis are not the problem (BTW you cannot get rid of Triton by dialysis, unless you use very high cut-off; although in your case - with 120 kDa protein, you can use high cut-off:). Thus I would focus on the other options and eventually consider another host.