Hello everyone,
I'm medical genetics master student.
CCHV NP was cloned into pGEX-6p-1 vector with BamHI and XhoI restriction sites using cloning primers 5'CGGGATCCATGGAAAACAAATCGA-3' forward and 5'-CCGCTCGAGTTAGATGATGTTGGCGC3' reverse.The accuracy of inserts was verified by sequencing.
After that, I did transformation and the recombinant plasmid was transformed into E.Coli strain BL21(DE3) and overexpressed as a GST fusion protein.
Then I did IPTG induction successfully. After that I centrifuged them and harvested cells were resuspended in lysis buffer... I have prepared lysis buffer myself. My lysis buffer containing 20mM Hepes (pH:6.8), 500mM NaCI, and 1ug/mL DNAseI and 33mg/mL RNAse and 1M 1uL DTT, 60uL Triton, 150uL PMSF, 3mL lysozyme.
My wash buffer was prepared with 20mM Hepes and 500 mM NaCI ph:6.8
My elution buffer was prepared with 0.0307g reduced glutathione and 10mL wash solution.
I lysed my pellet with lysis buffer on the ice and I did sonication 15x 15s. The lysate was centrifuged at 6000rpm for 1 hour at 4°C to remove cell debris. The supernatant was then loaded into a GST column overnight at 4°C with resin. After that I collected flow through. Then I washed the column with the wash buffer (3x 5 mL). Then I loaded elution buffer and I concentrated it at4000rpm x 2 hours. My results are below. My protein was 82kdA, but I didn't do protein purification.
I want to get a nucleoprotein. Can you help with this? I don't know what to do.
I didn't get any results. Finally, I changed my buffers. I used 150mM NaCI and 20mM HEPES ph.6.8 for lysis buffer and wash solution. I used reduced glutathione, but I haven't got any results in the elution part.
What should I do? What is your advice? For example: a new cloning vector? pET14b // N-ter his tag or pET28b ye Cter his tag? If you could share your ideas, I would be happy. Thank you.
If you want to look my results, respectively; 1. picture is high salt, 2. picture is low salt.
1-Uninduced
2-Induced
3-lysis pellet
4-lysis sup
5-flow trough
6-wash
7-elution 1
8-elution concentrate
It looks like your protein was induced but in an insoluble form. Most, if not all, of it was in the pellet. What little was in the supernatant may have been aggregated and did not stick to the affinity matrix. Try inducing expression at a lower temperature. Slowing down cell growth will sometimes help to improve the solubility of the induced protein.
Hi there,
Is 82kD the MW of the GST fusion or of your protein? The intense band in the lysate doesn't seem to be efficiently retained by the resin. As there seems to be GST in your elution samples (MW 26kD) it might be cleavage occured at some point and that your protein is in the Flow Through and Wash. Have you checked the resin after experiment in case your protein remained adsorbed?
Hi Duygu,
there are some points you should share to make sure you get a fruitful advice:
Induction: At which OD did you do the induction? What concentration of IPTG, temperatur and duration did you use?
Lysis&Lysis buffer: Why do you use 500mM NaCl (very high)? Lower Salt concentrations (100-150mM NaCl) should be more than enough to obtain efficient lysis! Check the protocol for lysozyme lysis. Did you check success of lysis with microscope?
Clearing of lysate: 6000rpm seems pretty low! I used to centrifuge my lysates at 45000g (SS34 rotor in sorval) or higher (SW28 in Beckman ultracentrifuge) to get rid of membrane vesicles which contain lots of proteases.
Binding to GSH-beads: Have you ever tried to simply let the lysate run over your column once and directly wash and elute the protein? I never incubated the GSH-resin over night, but simply let the lysate run through the column once or twice followed by immidiate washing and elution. It worked well for me with several different proteins.
Multimerisation/Aggregation: It could be that your protein is insoluble, but it could also be multimerisation to big aggregates. Is your protein of interest known to form dimers? If so, the dimerization of GST itself plus fusion partner might lead to aggregation.
Best,
Kai
Hi again;
INDUCTION:
My OD=06 and then i did induction and i used 1M, 1ml IPTG in 250ml sample for induction part...
Lysis&Lysis buffer:
I found 2 articles and they were used 500mM NaCI so i used 500mM NaCI, there is no any reason. But i tried also 150mM NaCI, but i havent any good result. I ll look lysis protocol again and No, i didnt check in microscope..
Clearing of lysate:
When i started to do experiment, i used 16000RPM at +4C and 45 min. But my centrifuge machine was broken i have to use 6000rpm at +4C and 1 hour. Im waiting service to centrifuge machine. I use Beckman.
Binding to GSH-beads:
No i havent tried to simply let the lysate run over my column once and directly wash and elute the protein.
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