Protein Expression - Science method

Agree with Michael, the pET System Manual is surely a good resource for a newer. Good luck.

Hi there,

These are strong constitutive promoters and terminators. Any combination should be OK... I have successfully expressed simultaneously 6 human proteins from genomic insertions using the following combinations: ProPGK1+terTDH1; ProTDH3+TerADH1; ProHHF2+TerSSA1; ProCCW12+TerENO1; ProTEF1+TerENO2; ProTEF2+TerPGK1. The most crucial point being to optimize sequences for expression in yeast.

I think this grid like shadows because of diffrence in borders lightening.

To refuse it you must select border removed region of interest in camera setting Or if not availabe adjust microscope condenser hight and pupil to have uniform light.

Well could be the non-structural protein has a temperature dependent interaction with something in your expression vector. NS1 is known to affect lipids while NS4 Modifies the ER.

Robert Adolf Brinzer Dear Robert I can use this NEB DNase for my purpose.

Could you please take a look at this product and let me know your idea? Already bought one DNase but was not good enough to do this experiment. Thanks in advance

Hello Innocent Agida

Are you using siRNA? If yes, then you are most likely to face this problem.

You need to consider protein stability which is highly variable. It may so happen that your target protein may be highly stable with a longer half-life. In such a case, you may consider choosing shRNA-mediated gene silencing method over siRNA. While making a choice, you will have to consider the length of the assay as well as the half-life of the target protein.

siRNAs are transiently expressed in cells, while shRNA is stably integrated into the host cell genome. As cells divide, the shRNA is passed on to daughter cells. Using lentiviral vectors for expression of shRNA, provides permanent knockdown without needing to transfect the cells multiple times.

The protein levels subsequently go down over time because the shRNA constantly keep suppressing mRNA.

So, if you are trying to knockdown the expression of protein that has a long half-life, then stable expression of shRNA may be required.

Best.

Some general techniques you can do to improve your western blots are:

- Run your gels at a lower voltage for longer to get straighter lanes/bands.

- Try a wet transfer if you are finding it hard to detect your protein.

- Try incubating your membrane with the antibody diluted either milk or BSA (some antibodies just image better in either BSA or Milk!)

- Incubate your antibody at 4 degrees overnight to ensure specific binding.

- Try a more sensitive ECL solution if you are struggling to image your protein (signalfire by Cell Signalling is excellent).

I haven't heard of this being a problem.

It won't be as clean as having all your variants in the same genomic locus but you can transfer your library into a lentiviral vector and infect at low MOI in order to get one integration per cell.

Hi there,

I guess you tagged the protein with 6His. Did you try WB for revelation? No difference in Commassie staining patterns doesn't mean no expression (it could be low expression). Making the difference between no and low expression is quite important: if no expression there might be a construct issue, if low expression then optimization may be considered.

The strongest correlation will be with the strength of the promoter in pretty much any system. A low copy number plasmid with a strong promoter will make more protein than a high copy number plasmid with a weak promoter. This is true in microbial (bacterial) cells. In mammalian cells, expression can be amplified by gene duplication, and in the case of methotrexate amplification to produce a stable cell line. In this case, the protein expression can be more dependent on gene copy number, but not linearly correlated, and promoter strength still has a strong influence. Always start with promoter strength.

Leran Mao

I think maybe it is better to check the size of your LC, HC in conditioned media before purification via WB. This way should help you to distinguish whether the problem is from expression or purification. (though I think you actually done it ?)

please update your progress, I think it is a really interesting question

thanks for your sharing

good luck

pRSET vectors don't have the lacI gene (lac repressor) like the pET-vectors. There could be a problem with leaky expression before induction, especially if the protein is toxic for the cells. pLysS might help for better repression of T7 transcription activity.

I highly recommend using a different IgG, in my case our ab recognized a portion that may have been internal (hydrophobic), the switch to another IgG that recognized a different peptide was the solution.

Also, try to run your samples with Guad for (solubilizing) mixed in them, your POI may be stuck and not running into the gels.

Taking together: it is highly recommanded to start with a test serie. That means to test different antibody dilutions and different DAB reaction times. It takes time to move several sections from one bath to the next one. 1 min reaction time in DAB is very ambitioned under this circumstance. So take a DAB reaction time which makes it comfortable to work with. You can interrupt the reaction for checking under the microscope any time. A negative control is helpful to get an impression of the unspecific background.

Normally, we screen by expression in 5 mL cultures (in 50 mL conical tubes) according to the Pichia Manual (although we also use pPICZalpha derivatives which came out after the original manual). We screen by enzyme assays (synthetic colorimetric substrates), but also by SDS-PAGE. Although I tend to discourage using zeocin when expressing, since I have to pay the bills, the students may add it at this step. We tried tagging with eGFP to see if proteins were retained in the vacuole before, but it did not seem particularly effective, probably because expression was quite low.

Excuse me, has someone got the answer？

Dear Kriti,

Did you validate your antibody before starting?

@Atia Hakim, another good option to avoid cell contamination is to harvest a nurished culture supernatant (secretome) from MSC, centrifuge 500g 10mins, and add SN to Hek293T. 2 days for culture SN would be enough (MSC starting density 70%, 8ml of medium per 10cm culture dish). Then just lyse Hek293T attached on the plate, this is the best option. I dont recommend trypsinizstion before RNA extraction. Good luck!

You might also try running separate, replicate gels, one for phospho- and one for total protein. Instead of stripping and reprobing.

Also, are you 100% confident in the specificity of the phospho antibodies? Is it possible the bands for pSTAT1 and/or pSTAT3 are really aomething else?

Protein expression tells you, how much protein you have. Protein activity tells you, how active the protein is.

Imagine you have workers supposed to dig a ditch. How many workers you have = protein expression. The more workers, the more work they do per time.

But rather than letting them work with bare hands :), you can give them shovels. This would be some activator, for example some allosteric activator or phosphorylation of *some* enzymes.

Or you can let them work without boots and thus inhibit their activity, because who would like to work without boots, right? This would be analogy of inhibition.

Other aspects that affect enzyme activity are for example temperature and pH.

Yes, to determine SIRT1's activity, you need some activity assay. There is surely something published. You would need to have acetylated protein and peptide and measure either removal of the acetate from the protein/peptide or formation of the O-acetyl-ADP-ribose (product), or decrease of NAD+ in the reaction.

There seem to be several activity kits available:

Basically, you translate your open reading frame into a protein sequence and then translate back into cDNA using high frequency codons. There are automated tools for this on the web, where you may define constraints like the restriction sites you need for cloning and manipulating your sequence, adding tags and stuff like Shine-Delgarno resp. Kozak Consensus elements, etc. . Then have your new cDNA synthesized. It never has been this easy.

geneart.com used to have such a nice tool on their website.

Mammalian proteins are often not expressed well in E. coli for several reasons:

Given these factors, even with codon optimization and confirmation of insert orientation, there can still be challenges in achieving efficient expression of mammalian proteins in E. coli. To overcome these limitations, alternative expression systems such as mammalian cell lines, insect cells, yeast, or in vitro translation systems can be considered, depending on the specific requirements of the protein and downstream applications...

Yes, it is possible for a cell to express a protein without mRNA expression. This can occur through a process known as translation-independent protein expression. This process involves the direct insertion of a protein into the membrane or organelle of a cell. This can be done through the use of transfection, which involves the introduction of a DNA molecule into the cell, or through the use of viral vectors, which can carry the protein into the cell. Both of these methods can lead to the expression of a protein without the need for mRNA expression.

Proteins can move within cells without being unlocalized. Examples are diffusion, facilitated diffusion, active transport, exocytosis, and endocytosis.

If you need to adjust the pH of your cell culture medium, it is best to use a buffer solution. A buffer solution is a solution that resists changes in pH. You can find buffer solutions that are specifically designed for cell culture.

There are a number of reasons why the distribution of GFP can be uneven in cells. The expression level of GFP can also affect its distribution. GFP is often more evenly distributed in non-dividing cells than in dividing cells.The presence of other proteins can also affect the distribution of GFP. GFP may be more likely to accumulate in areas of the cell that are exposed to certain chemicals or conditions.

This should work ok, but I'd include an insect specific protease inhibitor cocktail and then snap freeze fast, maybe with LN2. Controls are also , of course, a very good idea! Good Luck!

I already did those parameters by ELISA and reviewers need confirmation by Western analysis

I'd consider including an anion-exchange chromatography step. The highly charged RNA should bind tightly to such a column.

Hi, I have experienced this as well, and the problem is more than likely protein toxicity.

What may be happening is IPTG induction is actually killing 99.9% of your culture, and then as a rare event suppressor mutants emerge and continue outgrowth. This isn't obvious in liquid batch culture that is typically used for protein expression.

To test this, try pouring 0.5 mM IPTG + Kan plates. Grow your culture out to where you'd typically induce (0.6). Then, plate the cells onto both a Kan plate and an IPTG+Kan plate. If the protein is toxic, you'll get a lawn on the Kan plate and somewhere between a handful and a few hundred colonies on the IPTG plate, which would represent >99% of the cells being nonviable.

A growth curve can also be diagnostic of this - you'll see growth immediately cease after IPTG addition, and it'll take a few hours to recover, but it will eventually recover because of the suppressor mutants taking over the culture.

Are your inductions typically long i.e. overnight? Or just a few hours? How do the cultures do after induction - does the OD plateau quickly near 0.8-1.0? Or does it saturate at >2.5?

Agree that just because it's DNA-binding, that doesn't mean it can't be secreted too. But if you are sure that it is not secreted, necrosis-type cell is the only way I can think of for it to get into serum. Also, are you sure the protein is only expressed in monocytes?

As for your result, are you confident your protocol for making the cell lysate does enough to preserve protein stability? Cell lysates can be fickle in that regard.

Another potential issue are posttranslational modifications. If the secreted form has different PTMs than the intracellular one (which is common), this might affect antibody affinity.

Assuming the band you are looking at is really your protein of interest, and not an endogenous E. coli protein, then the most likely explanation is that the protein is not really folding correctly and being processed in E. coli (probably removing the end carrying the 6XHis tag since you can't purify anything).

Have you run an induced or control plasmid control to be sure the 30kd band you are looking at is really the protein of interest?

there is kits with coupled transcription/translation so you don't have to purify your RNA. Maybe RNAzol purified RNA can work.

Dear Tien Le

Sobstitution of Asp with GLu is quite conservative sobstitution and in the most of cases it does not affect conformation and stability of a protein but each protein has its own properties and it is possible that in some cases it may alter the protein stability or ability of the protein to bind a cofactor and therefore activity and stability. Expression yield is ofter direclty correlated with stability, since a poorly stable protein is degraded and result in lower expression.

If a PDB file is avaialble for you protein you can try to predict the effect of mutations using some software as Popmusic

or you can try also to verify it by comparing the thermal stability of the purified proteins (WT vs mutant) with DSF which is a simple and fast tecnique that you can easly run if you have in your lab an RT-PCR instrument.

best regards

Manuele

1. I've never done this kind of assay, but I saw most of paper that have done LysoIP use 3xHA-TMEM192.

2. I have no idea about that.

3. Maybe you can switch into Lamp2.

Thanks for the answers so far. Yes, our current protocol uses predominantly the miniprep kit from Promega. I have not used the maxiprep kit but if we decided to diagnose plasmid purity as our next step we will certainly take it into consideration.

The cells grown in 30C showed good plasmid concentration (300 ng/uL) but the A260/230 ratio was still questionable, and the protein expression did not improve in these newly made plasmids.

So far it does seems the plasmids did not transfect well. Last week we ran qPCR on the vector +/- insert and some of our vector + insert showed the same level of transcripts as the original, but the ones with questionable quality did not show good transcription. We're now re-transforming the cells from the PCR reaction since the last time the ampicilin stock was starting to go bad which might have resulted in the bad plasmids (although they did sequence well).

I would recommend fellow researchers to use qPCR to correct for some effects of transfection at least once if anything seems questionable.

Maybe you have a lot of cell lysis. Try checking for some other proteins in the extracellular serum

Not necessarily. The level of gene expression does not always directly correlate with the amount of protein produced. There are many factors that can affect protein expression, including post-transcriptional modifications, translation efficiency, protein stability, and degradation.

Hello Juthathip Jurutha

As cancer cells display unique characteristics in comparison to normal cells, you may take advantage of these differences to differentiate between normal and cancer cells.

For instance, Breast cancer cells may overexpress specific receptors which, when activated can initiate downstream signaling resulting in the expression of genes for cancer cell proliferation, growth, survival, migration, angiogenesis and other vital cell cycle pathways.

There are various types of breast cancer, some have hormone receptors like estrogen or progesterone (some have both) and are called ER+ or PR+ breast cancer respectively. Other most common receptors that are overexpressed in breast cancer cells are part of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, for example, EGFR and HER2 are overexpressed in approximately 40% and 25% of breast cancers respectively and are believed to be responsible for more aggressive tumor behavior and poor prognosis.

You may use antibodies against these receptors and check for the expression of the protein in both normal and cancer cells. You may use immunohistochemistry for confirming the expression and expression location of proteins in tissue sections. For cell lines, you may use immunocytochemistry (ICC) which is performed on sample of intact cells. ICC is a common laboratory assay that can confirm the expression and location of target peptides or protein antigens in the cell via specific combination of antibodies and target molecules. These bound antibodies can then be detected using several different methods. It will allow you to evaluate whether or not cells in a particular sample express the antigen in question. In cases where an immunopositive signal is found, ICC will allow you to determine which sub-cellular compartments are expressing the antigen.

There are two different immunocytochemistry assays:

1. Indirect ICC which mainly includes preparation and culture of cells, cell fixation, serum blocking, primary antibody incubation, labelled secondary antibody incubation, staining, result judgment and imaging.

2. Direct ICC in which only labelled primary antibody is used without the secondary antibody, and the other steps are the same as that followed in indirect immunocytochemistry.

Best.

Hi,

You can do qPCR to check the expression of the target genes.

We got WB800N here: https://lifescience-market.com/index.php?main_page=product_info&products_id=64199

Million thanks Pankaj Sharma & Robert Adolf Brinzer for the suggestions.

DEar Bu Gayun

i do not tihnk that cell freezing increase the protein expression level, since once the cells are frozen all the processes, ag protein expression are also frozen. This approach it is generally used as a long term biomass storage.

Is it possible that in some cases. it may improve the efficience of the cell lysis and protein extraction since freeze/thaw is, itelf a possible cell lysis approach.

Regaridng the GFP, i think that is not the protein amount but the fluorescence intensity that may change a little in function of the temperature and therefore the pellet it seems coloured.

best regards

Manuele

I also have the same problem at the moment. 3 terminal T's of the reverse Primer are not inserted into the final sequence. Everything else worked fine, no mutations were visible in the final sequencing. Did anyone find a way to prevent it from happening without having to buy new primers?

I'm thinking of two approaches: NGS of mRNA expression (to assess changes on transcription level) and differential proteomics (e.g., 2D-DIGE) to find changes on protein level

Hi Sir,

You can check gene expression at protein level using Human Protein Atlas (https://www.proteinatlas.org). Also, NCI Proteomic Data Commons can be explored (https://pdc.cancer.gov/pdc/)

It is possible that the expression of Cas9 in your stable expressing cell line could affect the expression of other proteins in the transduced cells. Cas9 is an enzyme that plays a central role in the CRISPR/Cas9 genome editing system. It recognizes and cuts specific sequences of DNA, and this process can potentially affect the expression of other proteins in the cell.

There are several potential mechanisms by which the expression of Cas9 could affect the expression of other proteins in transduced cells. For example, Cas9 might bind to and cleave the regulatory sequences of certain genes, disrupting the normal regulation of gene expression. Alternatively, Cas9 might cause DNA damage or DNA double-strand breaks that could affect the expression of other proteins in the cell.

It is also worth noting that the expression of Cas9 might be influenced by other factors in the cell, such as the presence of specific signaling pathways or transcription factors. Therefore, it is possible that the observed differences in protein expression between transduced and non-transduced cells could be due to the combined effects of Cas9 expression and other factors in the cell.

To determine the specific mechanism by which Cas9 expression is affecting the expression of other proteins in your stable expressing cell line, it may be necessary to conduct further experiments. This could include analyzing the expression of specific genes or proteins using techniques such as RT-qPCR or western blotting, or examining the effects of specific signaling pathways or transcription factors on Cas9 expression and protein expression.

Dear Willem Bintig

some years ago, have tried to do some attempt of production of a 15N labelled protein sample using the Bioexpress 6000 15N labelled medium

and the results monitored with a NMR instruments were encouraging.

Unfortunatelly, since we had a small free sample of that media (which as you can see is very expensive), we were not able to do many other tests.

Since the Expi293 media allow the cells to growth up to high OD, we did not use the Bioexpress6000 during the cell propagation and trasfection but we replaced the Expi293 unlabelled media with the labelled one the day1 after the transfection before addiction of the enhancers. In this way similarly to what is done for E.coli with the Marley approach, we removed the unlabelled protein produced into the media and we use all the labelled compounds for protein sintesis and not cell growth.

Sincerelly I'm not sure in the level of labelling that you can obtain with this strategy is compatible with mass spectrometry experiments but in case you have an antigen highly expressed in Expi293 and you have money for by that media, it could be a promising approach.

You can see more detail of the protocoll that i used and the spectra at the minute 8'20'' of the follwing video

presemt on my blog ProteoCool (proteoCool N°30)

good luck

Manuele

I agree with your idea trying another tag for western blotting. As for the activity of regulator, I have also used EMSA and SPR methods to compare the affinity between DNA and my regulators. However the results could not answer my phenotype difference question. Thanks again for your kind suggestion! I will try flag tag or other tag! :)

There are plasmids with a few different origins that can be used. For example the p15 origin of the pACYC plasmids is fully compatible with pBR origin. Also there are plasmids (although much less common) with a variety of other origins from resistance plasmids, F etc.

Alternatively you can design your own plasmid that has two expression cassettes on a single plasmid and not worry about it.

Lastly, if each plasmid has its unique drug resistance marker, you can maintain two plasmids with the same origin in one cell so long as you maintain selection for both.

I suppose because it is a polyclonal antibody you are getting some non-specific binding of it to other random proteins. Are you seeing any evidence for this exact antibody working more cleanly elsewhere (and not evidence from the manufacturer website)?

It is because the T7 promoter requires T7 polymerase to function (and not the normal E. coli polymerase). In the expression strains used for pET type plasmids such as BL21 (DE3) and similar, the gene for T7 polymerase is integrated in the host genome and expression of that gene is under control by a lac operator. So the addition of IPTG turns on expression of T7 polymerase and this in turn activates transcription from the T7 promoter of pET17 and other similar vectors.

Maybe I was not clear enough in my original post, but I always analyse the supernatants and pellets by SDS-PAGE after osmotic shock/lysozyme extraction and the protein is present in the pellet, I am just not sure how fix that.

That could very well be an issue. You might need to supplement the media with additional glycine and/or check for codon bias usage.

Thank u

@ Vikram Kumar,

Of course, it depends on your protein of interest, but some proteins are mainly in complexes with other proteins. If such a protein is over-expressed alone, it is not very stable and can aggregate.

We had examples in our lab where mammalian proteins, upon overexpression in E. coli, always had very specific "contamination" with a bacterial protein. Using mass spectrometry, we could find out that the overexpressed protein formed a complex with a bacterial homologue of a highly conserved protein, which turned out to be a binding partner of our protein. The fraction of overexpressed protein, which was not bound, simply was not soluble and could not be purified.

So if you know that your protein is "not happy" (i.e. very unstable) without a certain interaction partner, it makes sense to co-express that interaction partner.

Plasmids for mammalian expression use different organism specific promoters, a eukaryotic ribosomal binding site, an intron in the CDS of the gene of interest to avoid bacterial expression and to increase expression in the mammalian cells and a poly A tail after the stop codon to reduce mRNA degradation.

Hepatocytes in the liver express and secrete a great deal of protein, including serum albumin, which is the most abundant protein in blood plasma.

Manuele Martinelli Thank you sir

Hi

You can do that by adding a cleavage site of known protease between M and F, and after the expression, you can easily remove the M from your protein.

You can use the TEV protease that recognises the site (ENLYFQ/G-) and remove the ENLYFQ from the N-terminal of the protein. So if you added the ENLYFQ/G between the M and F, such as ( M-ENLYFQ/G-F), after the expression, treat your protein with TEV protease to remove M-ENLYFQ/ leaving your protein.

I hope this helps you.

Thank you so much for the detailed explanation. I'm not sure about our data type, but obviously, it's not gel-based proteomics. Our proteins are label-free. And yes since we used Maxquant for the analysis, Perseus was recommended to me for the statistics. So I want to compare LFQ intensities of female and male mice and see which proteins are differentially expressed. So which test should I use on Perseus?

I agree with John Hardy Lockhart that -80 is a good choice for long-term storage. I would also suggest portioning out your mRNA into multiple tubes to avoid multiple cycles of freeze/thaw. Also, it means if something happens to 1 tube (contamination, being dropped) you will have backup tubes.

Look also a possibility of protein degradation by proteasome

Thank you all of you for the suggestions. I am now trying to reflect on this.

The first I though of, were the primers (not working), so we doubled checked it by using the same primers on 3 different positive control cell lines, and they worked perfectly fine (in the same PCR reaction). The 3 PCR controls were the same cell lines we used as a control for the immunocytochemistry. So, in this case there's nice correlation (also in quantitative terms) between proteins and RNA (by PCR). For RNA, all the different cell lines have been extracted in the same way, by the same expert person and RNA quantification and quality (prior PCR) was very good. So, for me it's difficult to think that only the RNA of samples of interest were degraded and not those ones of controls. Regarding the primers design, I checked and saw several people used them in publication and, is it possible they are well design for a cell cline and not working in another one? AS well regarding the antibodies for immunos, does someone know or think that an antibody can recognise the 'right' protein epitope in a cell line and mis.recognise the same protein in another cell line? In other words, could the immuno signal be a complete artefact? Thank you!

Your cells look like OK, no worries please.

Hi Jessica Lee

This may not be the answer, but have you considered different PTMs of β-catenin?

It might also be easier to interpret if you include the molecular weight ladder in the image. How much is the difference between day 7 and 14?

Best of luck!

Sam

If "using E. coli BL21 (DE3) GroEL/ES cell for my protein expression by IPTG at 0.1 mM concentration at 18 degree Celsius temperature" is already a successful optimized condition being set up, then that means your target protein is a highly expressed one in E. coli, and low IPTG concentration (such as 0.1 mM final) and low temperature (18 C), together with GroEL/ES chaperonin, can slow down the protein expression rate and increase the protein solubility which often assure the correct folding of recombinant proteins.

Dear Vimar

as Annemarie Honegger already told you in generall the signal peptides are located at the N-terminus.

Before add a signal peptide to you protein sequence, you have to checi if it already contain a signal peptide or not.

you can do it by:

Each organism carry a spefic signal peptide therefore in case your protein already cotain a signal peptide but you would like to express it in a different orgamism (eg yeast protein in mammalian cells) you have to replace the signal peptide with one specifi for the expression host.

Ciao

Manuele

In my experience, it doesn't work

for increase expression

nucleotide sequence>fusion tag aminoacid seq>copy number

moderate copy vectors are the ones for majority of the proteins

8/17/22

Dear Saleh,

I was working w/ IPTG for protein expression some time ago and had a similar question about its stability. We obtained our IPTG from Sigma-Aldrich. I went on their website and found that IPTG is stable at room temperature for up to 30 days. You might try contacting your IPTG supplier. They probably have a technical support/resource group that can answer your question.

I hope this information helps you.

Bill Colonna Iowa State University, Ames, IA wcolonna@iastate.edu

Given that in pET21, both the lacI and the amp promoters diverge from the MCS, it appears unlikely that expression of your gene arises from read-through from the vector. However, if your cloned insert contains some sequence upstream of the coding sequence, it may contain a promoter that functions in E. coli.