Protein Expression - Science method

Park Sowon This looks like typical AI generated garbage. Many words and links, but no answer at all. It plagues and poisons human-to-human interactions and transmission of real lab experience.

Hello Akshaya Nagarajan,

The two most common methods for protein quantification are based on either mass spectrometry or immunological techniques like Western Blotting.

Western Blotting is often used for protein identification or relative quantification. It can also be used for absolute quantification if appropriate calibration standards are used.

Mass spectrometry based techniques offer superior data quality and reproducibility. It can achieve low limits of detection, provided that technical pitfalls, such as incomplete protein extraction, incomplete proteolysis, and artifactual protein modifications, are appropriately controlled and considered.

You may use selected reaction monitoring (SRM), also referred to as multiple reaction monitoring.

A Western blotting assay essentially depends on the specificity of the antibody used. In contrast, an SRM assay depends on multiple parameters, such as the retention time, the mass-to-charge ratio of the precursor ion and selected fragment ions of the targeted peptide, and the relative signal intensities of the detected fragment (transition) signals.

These values are then weighted and combined to derive a score that indicates the probability that the targeted peptide has been detected.

The paper attached below will be helpful.

Best.

Hi Can,

Howard Salis, 1st author of the manuscript I had linked in my prior response to the question 8 years ago, was at UCSF at the time his manuscript was published. The manuscript link to the prediction program is dead but he put it on a new server when he moved to Penn State.

I was using a prokaryotic GateWay Destination expression plasmid. The donor plasmid in which I had cloned the gene was primarily designed for recombining into eukaryotic expression vectors. Therefore, I added a prokaryotic Ribosome Binding Site (RBS or Shine-Dalgarno sequence) to the forward oligo to PCR and clone the gene I was expressing. Cloning was very straightforward (I actually had a high school student do it as a summer project). BUT it took almost 2 years to get expression (when I found Salis' program's new site). It correctly predicted that when transcribed, the RBS in the 5' leader sequence would form a very strong hairpin with an upstream NotI restriction site and flanking sequence (so lots of GCs in a row) in the original Donor plasmid. The hairpin sequestered the RBS, preventing the ribosome from assembling on it so translation was squelched. Eukaryotic ribosomes assemble at the mRNA cap and just plow their way to the Kozak site, secondary structure be damned. Prokaryotic ribosomes instead assemble on the mRNA on the RBS located directly before the initiating methionine, but only if the site is accessible.

Salis' program analyzed the inputted sequence and predicts expression levels but doesn't advise how to improve it. I used a separate program that maps RNA structure (I don't remember its name but there are certainly better programs now), and used intuition to manually tweak the sequence to eliminate secondary structure, and then tested it with Salis' program. I kept reiterating this process many times until I thought I had absolutely maxed predicted expression. I then repeated this for the initial 6xHis tag at the N-terminus because it too was predicted to form a very strong hairpin - that involved just changing some of the CAC codons to CAU, so pretty easy. I'm not sure if the His hairpin affected translation but as all the changes were incorporated into a single oligo, there was really no additional work.

The first time I tried the optimized vector, I got rid of all the desperation techniques I had tried - tight repression of toxic expressed proteins, medium formulations, temperature, inoculation protocols, fancy bugs. Also exotic purification techniques, protease inhibitors, and lysate formulations. Absolutely everything. Just 1 L of barebones LB on a shaker overnight and a simple lysate poured over a nickel column. Ended up with hundreds of milligrams of almost pure protein. I had *no* evidence of expression prior. So much protein that it overwhelmed the endogenous E. coli biotinylation system (I had included a C-tag for biotinylating the protein) so I had to add a 3rd plasmid to the system to express additional biotinylating enzyme (the 2nd plasmid expressed the 2 casein kinase subunits to phosphorylate the target expressed protein, so yeah, kinda complicated).

Quite long, but I hope this helps. I would certainly contact Howard Silas for advice - he's the expert. And I'm sure things have advanced significantly in the 8 years since this question was originally posted. https://www.researchgate.net/profile/Howard-Salis

Assuming you can select for both plasmids separately (different resistances) then it should be fine. Both will be induced by IPTG induction assuming the strain is expressing sufficient lacI protein (otherwise it will be constitutive).

Even if the plasmids have the same origin you can co-transform them but you need to have different resistance elements to maintain selection for both of them.

Mohammed Rafeeq Ali Thank you so much. i am trying to optimize with your suggestions as well.

You can see from the marker protein ladder the size range of proteins that are separated in the gel. If the marker dye is still on the gel after electrophoresis, smaller proteins will most likely be located at the dye front. If the dye has run off the gel, the smaller proteins will have run off and will be lost.

Small proteins may be lost during blotting if the blotting time is too long.

20 mutations in a small protein like that may either lead to misfolding and subsequent degradation or to the accumulation of rare codons that don't match the preferred codon usage anymore. You could try to use E. coli expression strains with inactivated proteases in the first case and strains with extra copies of genes for rare tRNAs in the second.

there may be a bacteriophage that is killing your E.coli. Perhaps check for contamination either in your E.coli storage or your culture medium.

Gary James Hunter Thanks for your suggestion. I have BL21 (DE3) so I will try expressing the protein in it. There is another alternative called Rosetta 2(DE3)pLacI. I am thinking of trying that too.

Hi Louis-Charles Béland

I do not have a positive control cell line which i could use in the FC experiment.

I haven't done WB either, but I am going for WB confirmation now.

Protein Guidance:

1-The tag acts as a directional signal to enter the protein into the endoplasmic reticulum or across cell membranes. 2-The cellular machinery responsible for transporting the protein recognizes this tag.

3-Ensuring the fusion process: The tag helps push the protein to penetrate the cell membrane or endoplasmic reticulum in the correct manner, ensuring its fusion with the membrane.

4-Maintaining the tertiary structure of the protein: The tag maintains the tertiary structure of the protein during the fusion process, ensuring the integrity of the final shape of the protein.

5-Targeting the protein to the appropriate location: The tag directs the protein to the appropriate location within the cell, whether the plasma membrane or other internal organelles.

In general, the N-terminal fusion tag GAMGSGIQRPTST plays a fundamental role in ensuring the transport of proteins across membranes.

Manuele Martinelli Hi Manuele, actually I'm doing immuocytochemistry on the mouse cells. The technique of antibody labelling that you mentioned sounds so cool. I will take a look. Thank you so much for the information.

Colony PCR is notoriously unreliable. It's easy to get false positives due to residual insert on the media surface & also easy to get false negatives due to incomplete lysis/too many cells. Pick a few colonies & streak onto a new plate of selective media. Then, do an overnight liquid culture under selection & a plasmid prep. You can't rush quality. It's possible for colonies to "drop" a plasmid if the antibiotic degrades or if you get satellites.

Good luck!

In eukaryotes primarily the first start codon will be used, otherwise every internal Met would serve to initiate a protein. So it probably doesn't matter whether or not you have an ATG at the start of your ORF when cloned in frame with the signal sequence.

However it sounds like the protein you intend to make is normally not secreted, there are many examples where non-secreted (cytoplasmic) proteins can not be secreted even if you add a signal sequence. So just be aware that this may or may not work for you.

Thank you so much Manuele Martinelli this was really good knowledge and I have a good and clear understanding on the expression of TLR4. Thank you so much. I also think using mammalian cells are the best for expressing TLR4.

Dear @Hyunjin Kim

As @Michael J. Benedik suggested the induction should be done in log phase of the respective cells growth in order to maximise the induction probability, as the ratio of number of viable cells will be very high in log phase compared to the remaining phases. And I personally believe, OD of 2.6 is fine when you inoculate adequate inoculum during subculture(for say, loop full), if you do very small inoculum transfer during subculture yes 2.5+ would be late log phase or near stationary phase.

Go back to the original antibody. If it is no longer supplied then ask abclonal where the original source of the antibody came from and contact that company to buy the one that works well

Thank you for the answer. I am using Pichia wildtype (Mut+).

You are correct. Ideally we should not use any antibiotics.

Try running the gel again and load way less of the vector PCR products. You have 5-10x too much product for the gel. That's why you are seeing smearing, and overly bright, curved bands.

And run it for a longer time at a lower voltage. Your ladder is a bit too smeared to be useful.

Good luck!

Choose antibodies that are specific for the HPV16 E6 and E7 proteins to avoid cross-reactivity with other proteins. Some antibodies are identified by a clone number which can indicate their specificity and performance in certain applications. The optimal concentration of the antibody for Western blot may vary, so it's important to follow the manufacturer's recommendations or perform titrations.

In total cell lysate you will get only "loose" proteins (and only some histones) while most of histone rather tightly bound to DNA and will be in precipitate after lysis and centrifugation. I would advise any of harsher methods, including HCl.

Creating glycerol stocks of Pichia pastoris is a common method for long-term storage of the yeast strain. Here's a general outline of the procedure:

Materials Needed:

Procedure:

Notes:

.

Dear Faiza Quddus

the induction protocol depends from your media AND culture volume.

If you are using LB in general you can growth an Overnight preculture (non inbduced), dilute in the morning it at least 1:100 in a fresh LB media carring antibiotics, growth the cells at 37°C up to OD(600nm) of about 0,6-0,8 and then induce with IPTG. THe lenght of the induction may change on the basis of induction temperature, eg. 3h at 37°C, 5h at 25°C or O/N at 17°C.

Lower temperatures may be usefull to improve protein solubility of stability.

you can find some information about in on the following link;:

best

Manuele

if "there's no promotor before my target gene"... then you have the answer

If there is no technical error, your result is normal.

You cannot make a correlation between the quantity of mRNA and the proteins produced.

The transcribed mRNAs are not automatically translated into proteins, there is what we call translational regulation and post-translational regulation, and these are 2 regulations which allow us to have a functional protein afterwards.

So even if an mRNA is present, it is not automatically translated into proteins.

A big issue is whether the protein is expressed in a soluble, soluble but aggregated, or insoluble form. If it is soluble but aggregated or insoluble, you may have a lot of trouble getting the protein correctly folded and in a soluble, unaggregated form for further work, if that is what you need.

If the protein has a tendency to be expressed in a soluble but aggregated or insoluble form, you might be able to change the expression construct to improve the situation, such as by making a fusion protein with a solubilizing protein or domain.

Also consider what method you will use for purification. Various affinity tags are available to help with purification.

The difference between using an IRES (Internal Ribosome Entry Site) and an IRES2 lies in their efficiency and specificity in driving gene expression in a bicistronic mRNA.IRES (Internal Ribosome Entry Site): IRES is a sequence element within the mRNA that allows ribosomes to initiate translation internally, bypassing the requirement for a 5' cap structure. When an IRES is present in a bicistronic mRNA, it enables translation initiation of the downstream gene even if the ribosome is still translating the upstream gene. However, IRES elements are generally less efficient than cap-dependent translation initiation, leading to lower expression levels of the downstream gene compared to the upstream gene.IRES2: IRES2 is an improved version of IRES that has been engineered to enhance its efficiency and specificity. IRES2 sequences have been optimized to increase translation initiation rates and reduce leaky scanning (initiation at inappropriate start codons). As a result, IRES2 elements typically lead to higher expression levels of the downstream gene compared to traditional IRES elements.In summary, while both IRES and IRES2 facilitate translation initiation of downstream genes in bicistronic mRNAs, IRES2 generally offers higher expression levels due to its improved efficiency and specificity.

Dear Sara,

In general the observation would be possible.

It depends on the duration of your experiment and how stable the mRNA and protein are.

You might have a temporary expression on an mRNA level and protein synthesis. If the protein is stable, mRNA-levels might be down already, while the protein is there.

Another explanation might be that the protein is usually burried somewhere in the nucleus and/or in protein complexes and that your treatment leads to a "release" of the (usually hidden) epitope.

If you have the chance, you could use a different pair of primers and a different antibody. That way you would at least rule out unspecific detection on mRNA and protein level.

Good luck,

Sebastian

Dear Zulfa Nasir

did you check the codon usage of your gene? Since in case of many rare codons you can try to otder and clone a sintetic DNA construct codon optimized. You can do it from Genescript, Geneart or other companies.

Moreover in case that expression is still low, you can try to add at the N-term an his tagged fusion tag a Trx,GB1 which are relatively small but able to push the expression level of the passenger protein and add the TEV cleavage site as a linker to remove the N-terminal tag after protein purification using a subctractive IMAC purification step.

you can find more informations about subctactive IMAC at the following link:

best

Manuele

Is there a marker protein you could blot for? You could then normalize the density of your signal of interest to that of the marker protein.

Dear Balakumaran Sathiyamani

Indeed, (in general) E. coli recombinant protein production systems are designed to achieve a high accumulation of soluble protein product in the cytosol. However, in the following excellent review:

Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in microbiology, 5, 172. https://www.frontiersin.org/articles/10.3389/fmicb.2014.00172/full

they rightfully state and I quote “Secretion to the periplasm or to the medium is sometimes the only way to produce a recombinant protein (Mergulhao et al., 2005; de Marco, 2009)… The signal peptides of the following proteins are widely used for secretion: Lpp, LamB, LTB, MalE, OmpA, OmpC, OmpF, OmpT, PelB, PhoA, PhoE, or SpA (Choi and Lee, 2004).” So, yes using PelB is worthwhile trying.

However, keep in mind that the selection the appropriate signal sequence is just one of the factors that determines the level of expression, see for example:

Jashandeep Kaur, Arbind Kumar, Jagdeep Kaur (2018) Strategies for optimization of heterologous protein expression in E. coli: Roadblocks and reinforcements. Int. J. Biol. Macromol. 106, 803-822 https://doi.org/10.1016/j.ijbiomac.2017.08.080

Best regards.

Robert Adolf Brinzer Thank you so much for your suggestion. can you share article to knockout gene?

Dear Stephan Spangenberg,

Thank you for your reminder! We have confirmed that the band size was due to non-specific binding. However, upon reevaluation using a highly sensitive chemiluminescent substrate, we successfully identified protein bands at the correct size.

Additionally, in the case of the splenocyte sample, after stripping and reprobing, I observed phosphorylated STAT1 (pSTAT1) but not total STAT1 protein. Interestingly, B cell samples appeared normal, and it is challenging to explain this phenomenon. To address this issue, I am considering running separate, replicate gels instead of stripping.

Thank you once more for your valuable input!

Hi all,

It's been a long time, to briefly update/wrap up on the project, we have used a higher pH in the buffer to condition our protein A column, and neutralized the antibody elution immediately upon collection with Tris buffer (pH 9). We also used another human cell line (COS7) for transient transfection (CHOS works okay but we observe more LC/HC fragments). With those changes we could collect more integrated antibodies detectably by Western blot and CE-SDS. We are not able to increase yield immensely but due to the nature of transient transfection (also no KOZAK sequence) this might be what we could achive at the moment.

We still have not figured out why we observed a 40kDa band intracellularly, but our antibodies were as expected post-secretion. It always seems the HC is problematic, and perhaps relates to its degradation. We used a HC-p2A-LC sequence and our LC can be expressed decently but not HC (intracellularly or extracellularly). Likely there is intracellular degradation for HC specifically.

Have you done three replicates of the same experiment and obtained the same results? Are the levels of the housekeeping gene similar in all the WB?

Dear Caio Braganca

a suggest to you to test the Brevibacillus expression sistem

i used in several years ago for expression of clostidium toxins

in parallel we tested also lactobacillus and Bacillus megaterium from Mobitec but i prefer Brevibacillus since B. megaterium is much more difficult to be transformed (you need to use protoplasts) and l.lactis due to the rapid media acidification do not reach high cell densites when you work in shake flask,

The main concern about Brevibacillus is that it smell quite a lot :-) but it is something that we can manage.

good luck

Manuele

Dear Haoran Z Du

in my experience the expression yields for large proteins (>100-150KDa) in E.coli is generally low, and often protein degradation in subdomain is observed due to the presence of proteases in he E.coli citoplasm and secretion ability of the E.coli in the periplasm is limited to lower MW (eg you can easly produce a Fab antibody in the periplasm while is very difficult to obtain a full lenght mab)

If you gene is derived from an eucariotic organism you can try to perform codon optimization to improve the expression yield but i think that mammalian cells as Expi293 are more able to work with so high MW.

best

Manuele

Your gel should have the exact same layout as your Western blot, because your Western blot is a transfer of your gel directly onto the membrane. You might not be noticing the right band on your gel. Maybe the band is very thin by eye with coomassie, or even barely visible, but it jumps out as a very major band with the antibody detection. This happens frequently. Most proteins studied are not the major protein expressed in the cell.

Do you have a different molecular weight ladder for Western and Coomassie stain? If you run the same ladder on both, that may clarify the situation.

Proteins do not necessarily run on the SDS PAGE gel according to their expected molecular weight from the protein sequence. There are several possible reasons for this, such as glycosylation and shape.

I would recommend a Western Blot analysis first in order to determine the concentration of the His-tagged protein in your lysate. Use a His-tagged protein with known concentration (if availble) to determine (roughly) your expression level.

These lateral flow tests are less sensitive than Western Blots and if you are using a cell line that is not normally used for protein production, the yields might be too low to get a good signal. Especially, if you are trying to optimize transfection conditions where differences of e.g. 20-50% more in one condition could hardly be detected.

I have produced and used similar His-tag lateral flow tests in the past and compared them to the ones from Thermo Scientific. Small differences are not easy to detect using this principle plus it is protein dependent (sometimes the tag is not well accessible and the result might be misleading). Western Blot is not exactly quantitative either but definitely more sensitive and not so much dependent on the structure of your protein.

Make cDNA from an RNA extraction and do a PCR.

No, it will not drive gene expression in bacteria since promoters work by recruiting specific proteins and the ones EF1alpha recruits are not expressed in bacteria, but in mammalian cells.

Hi

Usually, mRNA level does not have correlatation with its protein level. There are many factores that can interfer in this correlation. Usually, protein concentrations correlate with the corresponding mRNA levels by only 20 – 40%.

see: doi.org/10.1186/1471-2164-10-365

Hi Catherine, QIAGEN has a handbook for high-level expression and purification of 6xHis-tagged proteins. It helped me a lot when I was a master's student. It's available for free downloading at the following link

Hi Ross,

I have a similar problem. Did you solve the problem finally?

Hi Prakash,

The Santa Cruz Biotechnology, Inc. (https://www.scbt.com/home) company offers almost all ready-made shRNA or siRNA for known protein expressed in mammalian cells. You can search for your interested protein in HOME page. Some of their shRNA and siRNA used in our current and previous study is of good characteristics. I think this may be useful to your work.

Regards.

This review should at least et you started:

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

It is also possible that your gene is transcribed at a very low level but translated at a high level from those transcripts.

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.

As I am not certain what the product is that you may be working on, my response may be a bit more general.

Gene dosage is the number of copies of a particular gene present in a genome. Gene dosage is related to the amount of gene product (proteins or functional RNAs) the cell is able to express. Since a gene acts as a template, the number of templates in the cell contributes to the amount of gene product able to be produced.

Generally speaking, more copies of a gene — or higher gene dosage — will result in increased expression of the proteins for which the genes code. However, this is not always the case, as some genes are regulated by other factors that affect their expression levels. For example, some genes are dosage-sensitive, meaning that changes in their copy number can have significant phenotypic consequences, such as diseases or developmental defects.

An example of a dosage-sensitive gene is HBB, which codes for the beta-subunit of hemoglobin. Humans normally have two copies of this gene, one from each parent. However, some people inherit a mutated version of this gene that causes sickle cell anemia, a blood disorder that affects the shape and function of red blood cells. People who have one normal and one mutated copy of HBB are carriers of sickle cell anemia, and they produce half normal and half abnormal hemoglobin. People who have two mutated copies of HBB have sickle cell anemia, and they produce mostly abnormal hemoglobin. Therefore, the amount of protein expression from HBB depends on the gene dosage and the type of alleles inherited.

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!

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!