16 replies to this topic

[ram]

I have cloned 7 coding sequences from the plant in pEXP5 CT TOPO vector and right now trying to express them in BL21(DE3)pLysS. Expected molecular weight of the proteins is between 35 and 40 kD. All of them have proper reading frame and CT his-tag confirmed by sequencing. My problem is that out of these 7, only 1 is getting expressed as seen in the SDS-PAGE of the crude lysates and Ni-NTA fractions. There is no expression at all in rest 6! I am wondering why!! I have checked insoluble fractions also; they too doesn't have the protein that i expect!! I have reproduced the results 3 times. Twice with IPTG induction (2mM) and once with the Auto-induction system (Novagen). After picking up the colony from the plate, the cultures are throughout grown at 18C. The only construct that is working shows a huge recombinant protein band!! Can someone help?
Thanks

[HomeBrew]

Why the pLysS strain?  Do your plant genes that aren't working contain a lot of codons that are rare in E. coli?

[swanny]

HomeBrew, on Jun 25 2010, 12:06 PM, said:

Why the pLysS strain?  Do your plant genes that aren't working contain a lot of codons that are rare in E. coli?

pLysS is actually for toxic constructs, homebrew. You must be thinking of pRARE or Rosetta or one of those variants that code for the rare tRNAs.

Ok @ram, dumb question, but do each of the non-expressing clones have a start codon? I presume you've put the sequences into your program of choice and asked it to show you the expressed sequence.

[ram]

Thanks for replies!
@ HomeBrew: Because I also had read somewhere that pLysS has reduced levels of uninduced expression, so is good for expressing toxic proteins. I don't know whether my protein is toxic to E coli or not; but having a thought that it would be better than just BL21(DE3), I use pLysS.
@ Swanny: Yes! There are start codons in all of the plasmids. And the molecular weights that I have mentioned are from in silico translation in MEGA.

Edited by ram, 24 June 2010 - 10:12 PM.

[HomeBrew]

swanny, on Jun 25 2010, 01:10 AM, said:

pLysS is actually for toxic constructs, homebrew...

Yes, I knew that -- my questions (albeit crudely constructed) were actually on two unrelated topics.  Let me try again...

ram, on Jun 25 2010, 02:12 AM, said:

@ HomeBrew: Because I also had read somewhere that pLysS has reduced levels of uninduced expression, so is good for expressing toxic proteins. I don't know whether my protein is toxic to E coli or not; but having a thought that it would be better than just BL21(DE3), I use pLysS.

What you have said about pLysS is true -- the reason I asked you about it is because its presence suggested to me that you suspected that your gene(s) were toxic to E. coli.  So now my question is "do the cultures of the six clones that won't express grow normally (compared to the one clone that works) during your IPTG induction period, when the effects of pLysS are negated?"  More directly, since you suspected at one point that one or more of your genes might be toxic to E. coli, how have you now concluded they aren't?  Are BL21(DE3) cells without pLysS viable when transformed with your plasmids?

The second part of my original post was directed at seeing whether what was stopping your over-expression was an abundance of codons in your coding sequences that are rarely used in E. coli, to see whether one of the specialized E. coli strains containing pRARE (the Rosetta or Rosetta 2 strains, as swammy alludes to) might help overcome your expression problems.

Do the six clones that won't express contain a lot of codons that are rare in E. coli?

[ram]

Quote

Are BL21(DE3) cells without pLysS viable when transformed with your plasmids?

I did not check this. On the other hand, they are viable in Top10. But the toxic gene wont be toxic unless it is induced..right?

Quote

Do the six clones that won't express contain a lot of codons that are rare in E. coli?

Here is the number of rare codons:
Plasmid getting expressed:
arg 5 (AGG:2, AGA:2, CGA:1)
Leu 3 (CTA)
Ile 5 (ATA)
Pro 1 (CCC)

NOT getting expressed
Arg 9 to 10 (AGG:4 to 5, AGA:4, CGA:0 to 1)
Leu 2 to 5 (CTA)
Ile 2 to 6 (ATA)
Pro 2 to 4 (CCC)

Is this difference significant?

[HomeBrew]

ram, on Jun 27 2010, 07:48 AM, said:

I did not check this. On the other hand, they are viable in Top10. But the toxic gene wont be toxic unless it is induced..right?

Yes, but TOP10 cells don't have the T7 RNA polymerase necessary to express the gene in your pEXP5 CT TOPO clone, so the ability of these plasmids to be maintained in this strain gives no information about their toxicity.  It is true that a toxic gene cloned in a BL21(DE3) pLysS background will be stably maintained because the T7 lysozyme encoded by pLysS suppresses the chromosomally-encoded T7 RNA polymerase, but as soon as you add IPTG this suppression effect is overcome, and the cloned gene is expressed.  If this gene is toxic when expressed, the cells containing it will die under IPTG induction, and your ability to get over-expression of this protein from your IPTG-induced culture is nullified.

I suggest you try and determine definitively whether your cloned genes are toxic to E. coli when expressed by transforming them into a BL21(DE3) strain without pLysS.

ram, on Jun 27 2010, 07:48 AM, said:

Is this difference significant?

I don't know -- I'm uncertain how to decipher the information you posted.  What does this site have to say?  My understanding is that it's not only the absolute number of rare E. coli codons used by the foreign gene, but their placement in the coding sequence as well.  For example, you may be successful at expressing a gene containing four rare E. coli codons scattered throughout the gene, but be unsuccessful at expressing a gene with four rare E. coli codons in close proximity to one another.

[swanny]

... especially if they are clustered at the start of the coding sequence.

Have you done a timecourse of the expression? That is, does induction lead to death of the cells, or does the OD plateau?

[HomeBrew]

That essentially what I was asking a few posts ago, but the question wasn't answered:

HomeBrew, on Jun 25 2010, 07:17 AM, said:

...do the cultures of the six clones that won't express grow normally (compared to the one clone that works) during your IPTG induction period, when the effects of pLysS are negated?

[ram]

Thanks for replies and the suggestions HomeBrew and swanny. I will have deeper look at the sequences for the rare codons!
No, I did not see what happens to the growth after induction. May be I will observe it in the next experiment.
@ HomeBrew. I would have definitely liked to check the expression in BL21(DE3). But I don't have the cells right now I will see if I can get those from someone.

[shane]

GenWay’s recombinant protein production in E. coli provides services and solutions for antigen production, ligand preparation, and proteins for binding assays, cell based assays, enzymatic assays, vaccine development and therapeutics.

[ram]

Hi...I am back with the new results. I tried BL21 Star DE3 cells for expression. And with some modification, I could express all 5 remaining constructs! (for some reason, I left working with one). But now I have a new problem. In all these 5, my protein is in insoluble fraction! I did lot of literature searching for getting soluble protein, which forced me to do some variations in the experiments. This include, inducing below 0.5 OD, using lowest concentration of IPTG (0.1mM till 0.5mM), adding 3% ethanol just before induction, heat shock at 47C just before induction, including cofactor (Mg2+, in my case) in the media, using Novagen autoinduction media, using 2X LB-phosphate media, media with sorbitol and betaine. This, especially the last one, gave me some soluble protein, but it isn't sufficient for me, and I am seeing tons of protein in insoluble lane! Any other suggestion to convert this into the soluble form? I am somehow hesitating to denature the inclusion bodies; no guarantee of activity after refolding! Adding thioredoxin tag would be the another option, but it would require re-cloning!  
Thanks!

Edited by ram, 27 July 2010 - 07:19 AM.

[bhavana]

how did u proceeded with ur insoluble fraction for other six clones?

[ram]

Hi Bhavana
I didn't get your question
Anyways...I did't do anything with the insoluble fraction of any of the 5 remaining clones. I was just trying get the protein in the soluble form. Are you trying to ask how can the protein be solubalized?
As mentioned in my previous post, out of several changes made to get the soluble protein, those done using LB media having sorbitol and betaine succeeded to some extent and right now I am proceeding with those since I get at least some protein out of it

Edited by ram, 11 September 2010 - 04:27 PM.

[bhavana]

my question is that how did u know that the protein is present in insoluble fraction? I hope u added some chaotropic agents or denaturants to the pellete fraction ( inclusion bodies),what is the exact protocol u used to check the presence of protein in inclusion bodies.