Hello everyone,
I'm currently trying to overexpress and purify a human hormone that is a member of the TGF beta superfamily (536 a.a without first 24 a.a signal peptide) with 7 cysteine amino acids and 3 disulfide bonds in its last 108 amino acids(c-terminal). I'm currently using the pET28-a vector and T7 shuffle strain for expression in terrific broth (TB) media.
Expression protocol:
5ml overnight culture in LB Broth media.
50ml expression culture in TB media that inoculated 1:100 with overnight culture and incubated at 37 °C until OD 600nm: 0.9.
The culture was induced at OD 600nm: 0.9 with 0.3Mm and 50 uM of IPTG final concentrations and incubated in a shaker incubator at 15 °C temperature for 24 h.
Native and denaturing purification with Ni-NTA column protocol:
Nah2po4 50mM, Nacl 0.5M, for all solutions and 10mM of imidazole for Lysis buffer, 30mM, 60mM and 90mM of imidazole for wash buffers, and 500mM of imidazole for elution buffer. Also, for denaturing purification, I have made 8M urea with the above buffers. (pH=8)
My goal is to inject purified recombinant protein into mice to produce monoclonal antibodies against the recombinant protein. But I encountered results that I want to share with you and then ask my questions.
Results:
When I purified the expressed lysate with native Ni-NTA column protocol I got many bands in SDS-Page even with protease inhibitor cocktail (SIGMAFAST™ Protease Inhibitor Cocktail Tablets, EDTA-Free) but when I purified the expressed lysate with denaturing Ni-NTA column protocol I got an overexpressed almost single band in SDS-Page with my recombinant protein molecular weight(SDS-Page pictures are attached).
Also, I did gradient dialysis to remove urea, but about 80% of the recombinant protein turned into aggregate.
I tested the native and denaturing purified recombinant proteins in the commercial “Double Antibody Sandwich” Elisa kit and I got OD in 450nm which means two mab antibodies in the Elisa kit recognized the recombinant proteins.
Questions:
1. Is it a self-proteolysis when the recombinant protein has its 3D folding during native purification? And why it’s not happened when the recombinant protein is expressed in the cytoplasm of the bacteria?
2. Can I use the purified recombinant protein with urea for injections? Or until which concentration of urea in the recombinant protein is acceptable for injection?
Thank you for your time,
Best regards,
Farshid
Dear Farshid Moosavi
i suspect that your protein, due to the presence of the 3 S-S bonds is not expressed in the folded conformation in E.coli cytoplasm (which is a strong reducing enviroment that do not allow the formation of S-S bonds) and therefore once that you perform the cell lysis, the proteoases present in the E.coli soluble fraction are able to broke it. When you are performing extraction with UREA, those proteases are denatured by UREA and therefore your protein is more stable.
You can try to add proteases inhibitors but their stability during time is limited.
I think that you can try to express you protein in the E.coli periplasm (adding a pelB or ompA signal peptide at the N-term) as well as try to express it in Origami strain.
I suggest to use Enpresso B media instead TB. Is it more expensive but it show much better performances in my experience.
Regarding immunization in Urea and the maximal urea acceptable concentration, i think that it may differ in the different countries on the basis of the law that regulating the animal care.
However several years ago we were allowed to use samples were urea was replaced from 0,5M arginine, which is a strong agent and in many cases is able to mantain protein in solution with-out Urea. But i'm not sure if a so high Arginine concentration is still acceptable.
However immunization with unfolded proteins (as urea or arginine) may induce the production of antibodies recognizing linear but not confromational epitopes, those may work very well for some application, ad WB but may be less potent for others. It depends from the use of your final mabs.
I think that in theory the best alternative, is the expression in mammalian cells as Expi293 or ExpicHO but for this you need specific instrumentation (eg CO2 shaker incubators) and if you do not have this avaialvle, you have to externalize the production to companies as Genescript or Biontron and of course the cost may be higher that E.coli.
good luck
Manuele
You don't need a lot of the protein to inject into mice. So I suggest you refold the urea-denatured protein in the presence of 0.5 or 1 M arginine and with a glutathione redox buffer (typically 3 mM reduced glutathione, 1 mM oxidized glutathione), then pass it through a preparative gel filtration column equilibrated with PBS or other innocuous buffer to remove the aggregated protein, arginine, residual urea, and glutathione. Then you can safely inject the monomeric, refolded protein in the innocuous buffer. You may want to send it out for endotoxin testing first.
Hello, I follow the protocol described in this article https://pubmed.ncbi.nlm.nih.gov/28274805/ with minor modifications and it works great.
"When I purified the expressed lysate with native Ni-NTA column protocol I got many bands in SDS-Page...1. Is it a self-proteolysis". Those bands may just be contaminating protein bands and not necessarily indicate proteolysis. Under denaturation conditions in Ni-NTA non-specific binding from those contaminating proteins is reduced while your protein of interest containing a synthetic continuous stretch of his-tag is still capable of binding. Tweaking purification conditions (washing column longer post protein binding, employing a narrow range of imidazole gradient for elution, adjusting imidazole concentration in sample buffer) may therefore help increase protein purity in native purification.
"Also, I did gradient dialysis to remove urea, but about 80% of the recombinant protein turned into aggregate." You would need to avoid large pH jumps during dialysis. If the difference in pH of your elution buffer and final buffer is huge with the pI of your protein of interest falling in between this range, aggregation may be encountered. In such cases, you would need to remove urea first without changing the pH of your dialysis buffer and then do step dialysis towards your desired pH to enable the folding of your protein of interest. This may rescue your protein from observed aggregation.
Farshid Moosavi
I hope the above information helps.
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