Hi guys. I am currently trying to express a hypothetical protein that belongs to Mtb in an e.coli BL21 host cloned into a PGEX vector. Upon bioinformatic analysis it shows that this is an integral membrane protein. I am going to express using IPTG for 3 hours at 37 degrees w/ shaking. The protein does not appear upon sds analysis ( i pellet it after 3 hours and re-suspend in PBS- thereafter I add sds loading buffer (1:1 ratio) and heat to 90 degrees before loading on gel). Are there alternate protocols for extracting an integral membrane protein? Thanks in advance
Several protocols are available. One problem in your prep is you are trying to heat membrane protein which is not recommended in preliminary trial and error steps - just use room temperature and check it.
Hello Diya,
How do you know that your protein is absent ? From the protocol you describe, what you load on the gel is a total extract of all E. coli proteins and it may well be that your protein is masked in the whole set of hosts proteins. Is an antibody available to check for the presence of the target polypeptide by Western blotting ? Therre are many reasons why a recombinant protein could be poorly produced: mRNA instability, poor codon usage, protein degradation etc. I don't see what is wrong with your extraction procedure: heating a membrane protein in SDS is no no more likely to affect its covalent structure than it will for any cytoplasmic protein: in both cases the protein will be denatured, but the polypeptide chain will remain intact.
Hi Pierre Béguin
Thank you for your response! Upon analysis of the SDS gel ( please see attached) I do not see a difference between the induced ( first six lanes) vs uninduced or in the lane which contains just pGEX ( shown in the second last lane next to the protein marker) I have tried western blot using anti-GST as my primary antibody and anti-mouse as my secondary. Unfortunately I did not obtain any signal upon staining with TEMED.
Hello Diya,
If you have the reagents to perform Western detection of GST, you should be able to see the GST band in the induced pGEX control; if not, there is something wrong with the Western blot protocol/reagents. If there is a good detection of the GST band with the pGEX control and none with the recombinant plasmid, then the recombinant protein is not detectably produced. Regarding the gel that you show in your response, not knowing exactly which lane is what, nor the expected size of the target polypeptide, it is difficult to make an opinion, but it seems to me that there is a band slightly slower than the darkest marker band (50 kDa ?) that is clearly present in lanes 3-5 of the gel and absent in the two rightmost lanes (controls ?)
Hi Pierre Béguin
Sorry I forgot to include that the expected size of the protein is 32.5 kDa with the GST tag, without it is around 5.7 kDa. With regards to the gel ( from the left to right) the first lane to the sixth lane is induced. With lane 1 being T1 ( hours after induction)
lane 2 - T2
lane 3- T3
lane 4- T4
lane 5- T5
lane 6- T overnight
lane 7- uninduced
lane 8- Pgex control
lane 9 - Protein marker
With regards to my western blot I believe I overly diluted the primary antibody ( used 1:5000 instead of 1:1000) so will re-do that and hopefully will detect even Just the GST. I really appreciate your feedback :)
If the size of third marker from the bottom is about 30 kDa, it seems to me that there is a band of about the right size that is clearly becoming stronger bstween lanes 1-6, and is nearly absent in the control lanes 7 and 8. However, further confirmation by western blotting or mass spectrometry would be advisable, unless the polypeptide also carries a His tag, which would enable its purification (GST is useless after denaturation with the SDS sample buffer)
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