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No activity of fungi protein in E.coli

heteroexpression

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15 replies to this topic

#1 Biogareth

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Posted 13 October 2011 - 12:30 PM

Hi all,
Recently I heteroexpress an enzyme from fungi in E.coli XL10Gold at 37 degree. After checking the activity, there is no activity at all, so do you think all protein expressed is insoluble?
It is supposed to be a little activity present even if the condition is optimal.
Could you give me some suggestions? Thanks so much!

Biogareth

#2 protolder

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Posted 13 October 2011 - 10:16 PM

Hola, this strain is a subclonig one for big DNA inserts and with white blue selection of positive clones. So, why don´t you isolate pure plasmid and try transform any expression strain as some of the BL21 family. Check phenotypes to select the most adequated for your construction Buena suerte

#3 Biogareth

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Posted 14 October 2011 - 12:16 AM

Hola, this strain is a subclonig one for big DNA inserts and with white blue selection of positive clones. So, why don´t you isolate pure plasmid and try transform any expression strain as some of the BL21 family. Check phenotypes to select the most adequated for your construction Buena suerte


Thank you for your suggestions!
I am wondering BL21 can accept the constitutive promoter instead of T7 promoter?
I also think that the temperature for incubation is quite important probably. How do you think?

#4 protolder

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Posted 14 October 2011 - 12:53 AM

Hola, I think that Bl21 without (DE3) coudl serve , but wait more advises of other forers . About temperature, for constitutive promoters I thing it hasn´t relevance but always more slow grow helps folding ofthe new sintetized protein at least in inducibles constructions. Buene suerte

#5 allynspear

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Posted 14 October 2011 - 06:00 AM

What promoter are you using? It is possible that your promoter is too strong and producing too much protein for the bacteria to handle. Also, constituative promoters are rarely a good idea without some good reason, especially for expressing foreign enzymes. The enzymes often do damage to the cell and cause the E. Coli to form inclusion bodies. It is much better to use an inducible system like the T7/BL21 expression like protolder suggested, since your bacteria will be fine until you induce them.

Lastly, the temperature issue is a tricky one. If you are producing a toxic enzyme, it won't matter what temperature you grow the cells at, they will eventually die when enough protein has been made. If your problem is protein folding, lowering the temperature can help produce the protein at a slower rate, giving the bacteria enough time to fold more of it correctly. Without knowing what enzyme you are expressing, I can't guess if it would be toxic to the cell, but there are ways to test if your protein is insoluable using Sonication (or other physical lysis proceedure) and SDS-PAGE.

(1) Take a 50 uL sample of whol cell suspension and add 50 uL of 2X SDS-PAGE sample buffer. Boil for 5 min.
(2) Sonicate ~10 mL of cell suspension using your favorite protocol (but keep on ice).
(3) Take 50 uL of the sonicated suspension and centrifuge at MAX speed in microfuge for 10 min
(4) Remove the supernatant from the centrifuged sample to a new tube.
(5) Add 50 uL of 2X SDS-PAGE sample buffer to both the pellet and to the supernatant tubes. Boil for 5 min.
(6) Run SDS-PAGE on all of your samples.

Your protein should always be present in the whole cell suspension (step 1), but if your sample is present in your supernatant sample then it is soluable, if it is in the pellet fraction it is insoluable. Like I said, this will only tell you if your protein is soluable or insoluable, but not WHY. It could be insoluable because of protein folding OR because of toxicity. If you can't see your protein at all, then your protein could be so toxic that the only bacteria that grow are somehow shutting down protein expression. Either that, or they have mutated your plasmid to block expression, I would definitely take some culture and grow up a small plasmid mini-prep to verify your plasmid sequence, both your gene AND the promoter region.

Best of Luck.

#6 Biogareth

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Posted 14 October 2011 - 12:13 PM

What promoter are you using? It is possible that your promoter is too strong and producing too much protein for the bacteria to handle. Also, constituative promoters are rarely a good idea without some good reason, especially for expressing foreign enzymes. The enzymes often do damage to the cell and cause the E. Coli to form inclusion bodies. It is much better to use an inducible system like the T7/BL21 expression like protolder suggested, since your bacteria will be fine until you induce them.

Lastly, the temperature issue is a tricky one. If you are producing a toxic enzyme, it won't matter what temperature you grow the cells at, they will eventually die when enough protein has been made. If your problem is protein folding, lowering the temperature can help produce the protein at a slower rate, giving the bacteria enough time to fold more of it correctly. Without knowing what enzyme you are expressing, I can't guess if it would be toxic to the cell, but there are ways to test if your protein is insoluable using Sonication (or other physical lysis proceedure) and SDS-PAGE.

(1) Take a 50 uL sample of whol cell suspension and add 50 uL of 2X SDS-PAGE sample buffer. Boil for 5 min.
(2) Sonicate ~10 mL of cell suspension using your favorite protocol (but keep on ice).
(3) Take 50 uL of the sonicated suspension and centrifuge at MAX speed in microfuge for 10 min
(4) Remove the supernatant from the centrifuged sample to a new tube.
(5) Add 50 uL of 2X SDS-PAGE sample buffer to both the pellet and to the supernatant tubes. Boil for 5 min.
(6) Run SDS-PAGE on all of your samples.

Your protein should always be present in the whole cell suspension (step 1), but if your sample is present in your supernatant sample then it is soluable, if it is in the pellet fraction it is insoluable. Like I said, this will only tell you if your protein is soluable or insoluable, but not WHY. It could be insoluable because of protein folding OR because of toxicity. If you can't see your protein at all, then your protein could be so toxic that the only bacteria that grow are somehow shutting down protein expression. Either that, or they have mutated your plasmid to block expression, I would definitely take some culture and grow up a small plasmid mini-prep to verify your plasmid sequence, both your gene AND the promoter region.

Best of Luck.



Hello allynspear,

Thank you so much for your suggestions.
First of all, I don't think this protein I want to expressed is not toxic, because this enzyme expression in E.coli was already publised by one Japanese group. So probably it it the problem of protein folding for my case. But when I checked insoluble and soluble proteins on SDS-page, I found the target proteins is equally distributed in two fractions.How do you think?
BTW, my gene sequence was already confirmed by sequencing.


#7 allynspear

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Posted 18 October 2011 - 07:07 AM

Well, the fact that half of your protein is insoluable does make me lean towards a folding problem, which is where I would suggest growing at a lower temperature to see if that gives you an active enzyme. However, with half of the protein being soluable, it is a little strange that you see zero activity. It is possible that there are inhibitors of this enzyme either in the E. coli or in the lysis buffer that you are using.

Are you following the exact protocol from the Japanese group that published before? Sometimes things that look minor in a protocol can turn out to make a big difference in activity. If you could post a link to the previous protocol or at least indicate what your enzyme assay is, I might be able to make some suggestions.

Best of Luck.

#8 Biogareth

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Posted 19 October 2011 - 06:35 AM

Well, the fact that half of your protein is insoluable does make me lean towards a folding problem, which is where I would suggest growing at a lower temperature to see if that gives you an active enzyme. However, with half of the protein being soluable, it is a little strange that you see zero activity. It is possible that there are inhibitors of this enzyme either in the E. coli or in the lysis buffer that you are using. Are you following the exact protocol from the Japanese group that published before? Sometimes things that look minor in a protocol can turn out to make a big difference in activity. If you could post a link to the previous protocol or at least indicate what your enzyme assay is, I might be able to make some suggestions. Best of Luck.



Thank you allynspear!
I posted the protocol for expression of enzyme and enzymatic assay as follows:
A portion (10 ml) of seed culture of E. coli BL21 (DE3) harboring the recombinant pET plasmid was inoculated in 1 l of Luria-Bertani (LB) broth supplemented with 50 lg/ml ampicillin and grown at 30 °C overnight. The cells were washed once with 10 mM TRIS/HCl (pH 7.5) containing 150 mM NaCl and suspended in 20 mM potassium phosphate bu€er (pH 7.0) containing 20% (v/v) glycerol, and 1 lM each of pepstatin A and 4-(2-aminoethyl)-benzosulfonyluoride hydrochloride. The cells were disrupted by sonication and insoluble materials were removed by centrifugation. Fractions containing TSase were pooled and dialyzed against 20 mM potassium phosphate buffer containing 20% glycerol.
Enzymatic assay: was monitored by the change in absorbance at 340 nm, because the rate of NADPH
generation was proportional to the activity. The reaction mixture contained 200 mM trehalose, 40 mM potassium phosphate
buffer (pH 7.0), 10 mM glutathione, 0.16 mM EDTA, 1 mM NADP+, 1.3 mM MgCl2, 0.067 mM glucose 1,6-bisphosphate, 1.55 U/ml PGM, 1.75 U/ml G6PDH, and an enzyme sample in a total volume of 2 ml.

But for my experiment, I did same modification for their protocol. 1) We don't have the pET plasmid with inducible promoter, therefore, I used constitutive promoter for protein expression in BL21; 2) We didn't add 20% glycerol as stabilier normally, because there is no difference when we added it in the solution; 3) Enzymatic assay also has some modificaiton for concentration of composition, but it should be no problem; 4)For the protein expression, I only check the crude enzyme by SDSpage at 90KDa. Do you think I have to do western blog to confirm the protein expression.

BTW, my gene is codon optimized in E.coli, do you think folding problem is still present?
Thanks!

Biogareth

Edited by Biogareth, 19 October 2011 - 06:44 AM.


#9 allynspear

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Posted 19 October 2011 - 01:05 PM

I am assuming that you are just using your cleared, sonicated cell-free extract for your enzyme assay and not fractions collected by purification. When you say there was no difference with or without 20% glycerol, what do you mean? no difference in purification or activity?

To check your expression by SDS-PAGE and Staining alone, you have to have some control lane. Do you have cells tranformed with the empty vector that you can compare to, showing that you don't see that 90 kDa band without your expression plasmid?

Lastly, codon optimization is actually just a way to give you more full length protein, not necessarily for folding. A codon optimized gene will be translated faster and to higher levels than a gene with sub-optimal or rare codons. So even a codon-optimized gene can have folding problems.

The enzyme assay for TSase required other enzymatic steps (i.e. PGM & G6PDH), so I hope you have a positive control for the assay to be sure your reporter enzymes are still working.

#10 Biogareth

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Posted 19 October 2011 - 11:50 PM

I am assuming that you are just using your cleared, sonicated cell-free extract for your enzyme assay and not fractions collected by purification. When you say there was no difference with or without 20% glycerol, what do you mean? no difference in purification or activity?

To check your expression by SDS-PAGE and Staining alone, you have to have some control lane. Do you have cells tranformed with the empty vector that you can compare to, showing that you don't see that 90 kDa band without your expression plasmid?

Lastly, codon optimization is actually just a way to give you more full length protein, not necessarily for folding. A codon optimized gene will be translated faster and to higher levels than a gene with sub-optimal or rare codons. So even a codon-optimized gene can have folding problems.

The enzyme assay for TSase required other enzymatic steps (i.e. PGM & G6PDH), so I hope you have a positive control for the assay to be sure your reporter enzymes are still working.



Thank you!
I didn't do the purification, and I used lysozyme to break the cell for receiving cell-free extract instead of sonication. There is no difference on activity (no activity at all) with or without glycerol, so I didn't add glycerol in later experiment.
For SDSpage, I didn't add control like same cells with empty vector, but I am wondering it will see the difference, because there are many crowded bands for crude enzyme on SDS-page.
For enzyme assay, I did the standard sample with reporter enzymes, and they are active.

#11 allynspear

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Posted 20 October 2011 - 04:15 AM

Without some control to verify expression, you can't be sure that you are actually expressing enough protein to see activity. If you cant be sure that the band you see is your protein, and you have no activity by enzyme assay, then you either need to have an empty vector protein extract control or do a western blot for TSase. I know that there are commercial antibodies available for TSase, but not sure if they are for the same species as you are working with.

If the other group that you were working with was successful using the pET vector system, and you are having trouble with a constituative promoter, it is possible that your protein expression levels are not high enough or that your protein concentration in the crude extract is not high enough to give detectable activity. The pET system produces very, very high levels of protein in a short time, in a smaller number of cells. You could just be having a concentration issue, especially if you cant directly observe your band in an SDS-PAGE. Most proteins expressed in the pET system will show up as a huge blob on SDS-PAGE of crude extract.

Lastly, did the other group show activity in crude extracts? If not, it is possible that there are inhibitors present in the crude extract that were removed during the fractionation performed by the other group.

Best of Luck.

#12 Biogareth

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Posted 19 November 2011 - 05:10 AM

Without some control to verify expression, you can't be sure that you are actually expressing enough protein to see activity. If you cant be sure that the band you see is your protein, and you have no activity by enzyme assay, then you either need to have an empty vector protein extract control or do a western blot for TSase. I know that there are commercial antibodies available for TSase, but not sure if they are for the same species as you are working with.

If the other group that you were working with was successful using the pET vector system, and you are having trouble with a constituative promoter, it is possible that your protein expression levels are not high enough or that your protein concentration in the crude extract is not high enough to give detectable activity. The pET system produces very, very high levels of protein in a short time, in a smaller number of cells. You could just be having a concentration issue, especially if you cant directly observe your band in an SDS-PAGE. Most proteins expressed in the pET system will show up as a huge blob on SDS-PAGE of crude extract.

Lastly, did the other group show activity in crude extracts? If not, it is possible that there are inhibitors present in the crude extract that were removed during the fractionation performed by the other group.

Best of Luck.



Hello allynspear,

I am still focusing on this problem. Recently, I tried to express this protein with a strongest constitutive promoter we have, and cultivate them under different temp (25, 30, 37 degree), but there is no activity at all. We tried western blot to check His-tag protein in pellet and supernatant, but no band is present. It is really strange. There is no reason that it can not express any proteins. I will re-do western blot to confirm it.
Additionally, I transfer my gene into pTrc vector (not pET vector). I suspect that protein expression is so dependent on the promoter, even I just want to see a little activity first.

Biogareth

#13 pDNA

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Posted 19 November 2011 - 07:58 AM

Dear Biogareth,

can you give us the link to the Japanse paper you are referring to? ...i would like to know what pET vector they are using?!?!?!

Maybe your protein contains disulfide bonds and does not fold correctly in the cytoplasm and was therefore secreted to the periplasm by the Japanese people to allow for proper folding?

Regards,
p

#14 Biogareth

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Posted 19 November 2011 - 10:34 AM

Dear Biogareth,

can you give us the link to the Japanse paper you are referring to? ...i would like to know what pET vector they are using?!?!?!

Maybe your protein contains disulfide bonds and does not fold correctly in the cytoplasm and was therefore secreted to the periplasm by the Japanese people to allow for proper folding?

Regards,
p

The link:
http://www.ncbi.nlm..../pubmed/9763690

#15 pDNA

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Posted 19 November 2011 - 02:50 PM

pET16b has no leader for secretion ...so we can rule that problem out!

Have you ever checked plasmid stability? ...maybe, since you use a constitutive promoter, there is strong selection pressure towards plasmid free cells or mutations that abolish production of your gene of interest. I would use an inducible system (and stick to the protocol as much as possible!).

Another thing is that they assayed the activity after purification with a Ni-column ...maybe your enzyme is not active in the crude extract? ...or did i got something wrong and you also purified your enzyme?

Regards,
p




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