Questions tagged [protein-expression]

The process by which proteins, encoded in the genome, are ultimately synthesized.

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2answers
52 views

Why does it matter to predict protein structure?

And how do you predict it ? What is your input data (sequence of amino acids, temperature, pH, ...) ? Is there a "standardized" input that scientists agree on ? Moreover, I've read that knowing the ...
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4answers
8k views

What is the longest-lasting protein in a human body?

Protein life times are, on average, not particularly long, on a human life timescale. I was wondering, how old is the oldest protein in a human body? Just to clarify, I mean in terms of seconds/...
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1answer
91 views

Centrifuge after sonication

I follow a protocol to get protein from E.coli cells after sonication. I used to grow 6 litres of large cultures and add IPTG to express the protein. I centrifuge for 10 mins at 8,000 rpm and get the ...
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0answers
44 views

How are animal patterns encoded in the dna?

After seeing the patterns on the feathers of a argusianus argus pheasant (shown below), I am curious where is the information that encodes a pattern for a particular bird, and what form is this ...
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2answers
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How useful are vitamin and trace element additions in E. coli culture medium?

In protocols for minimal media for growing bacteria I often see that a vitamin mix or a mix of various trace metals is added. But at least for E. coli I know that those are not strictly necessary, ...
2
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1answer
196 views

How can carnitine enter the mitochondrial matrix without OCTN2?

Some people have defects on the gene SLC22A5, giving them problems with their OCTN2 transport protein (Organic cation transport). OCTN2 transports carnitine into the mitochondial matrix where it can ...
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0answers
35 views

How can I render a protein non-functional to avoid problem during expression?

We're expressing a protein in a model organism for crystallization, however it seems the protein has some toxic effect on the cells. Is there something we can do to make it non-functional during ...
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0answers
62 views

Protein bands not visible after sds page coomassie dye stain

I am working with cell wall protein of Candida albicans and cryptococcus neoformans but after protein extraction and run on SDS PAGE my bands are not visible after coomassie staining. Can anyone tell ...
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36 views

Has network theory been successfully applied to protein expression within a cell?

I saw the following passage in the NPR 13.7 Cosmos & Culture commentary post The Big Idea Behind Big Data. Using the Internet's first generation of Big Data, researchers like physicist Albert-...
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1answer
142 views

How do I improve expression using the GAL1 promoter in S. cerevisiae above the “leaky” level I'm currently seeing?

I am attempting to express GFP in S. cerevisiae using the GAL1 promoter. I always grow an uninduced (in dextrose based SD medium) culture alongside my induced (in galactose based SD medium) culture. ...
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1answer
690 views

Life cycle of proteins

I try to get a picture of the life cycle of a protein (considered as a specific molecule). This is how I can imagine it: After the cell is born a protein molecule is synthesized by gene expression ...
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2answers
121 views

Examples of heterologous expression in human cell beside viral expression

Usually, people are interested in expressing human proteins in fast-growing organisms like bacteria to yield a maximum of protein within a short time lapse. Codon usage harmonization/optimization have ...
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0answers
30 views

Expressing bacterial proteins in mammalian cells

Does anyone have experience/knowledge of expressing bacterial (or other microbial) proteins in mammalian cells? I am interested in this for a project where we are trying to introduce a new enzymatic ...
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1answer
39 views

What are the advantages using SUMO vector for expression? [closed]

My question is : What are the advantages using SUMO vector for expression?
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1answer
627 views

Is protein production doubled if you have homozygous dominant genes as opposed to heterozygous genes?

At school we were taught in quite simplistic terms. We were told that there is a dominant and recessive type of every gene and that if the dominant gene was present, the protein is produced and if it ...
5
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1answer
143 views

Optimization of Signal Peptides

We are looking at modifying the signal peptide of a receptor in a common immortalized human cell line. The cell line already expresses an unusually high amount of the protein, but much of it is not ...
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1answer
387 views

why do chaperones bring protein into mitochondria? why would mitochondria need protein?

Sounds trivial? Please help to sort out. I saw this picture while looking at dehydration reactions and cell revision. And proceeded to the reference web page. Protein Folding and Processing in ...
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0answers
57 views

Protein expression in K. lactis system

I am trying to express a protein in K. lactis Protein Expression Kit (E1000S - NEB) in the way that the protein is secreted into the medium. I know that I can get some protein but it is not active. As ...
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0answers
26 views

Do exon deletions in C.elegans mutants necessarily result in nonsense mediated decay of the transcript?

I am trying to write out my research proposal about a project in C.elegans. There are multiple mutants of the factor that I want to study, each with different mutations. I know that a mutation that ...
2
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1answer
346 views

If bortezomib, a cancer drug, inhibits cell proteasomes, wouldn't resulting protein aggregate in normal cells further increase the risk of cancer?

Bortezomib is an anti-cancer drug that inhibits the proteasomes of cancer cells, allowing proteins that stop cell growth to fold and perform their function. However, wouldn't bortezomib also affect ...
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2answers
335 views

Are there any examples in nature of two polypeptides joining into a single, continuous, third polypeptide?

Are there any examples in nature of two polypeptides join into a single, continuous, third polypeptide like this: (Where all the indicated amino and carboxyl groups are on the main polypeptide ...
2
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2answers
568 views

Protein denaturation and edible toxins

As a protein denatured, it may lose its functions. So theoretically if I heat some type of biological toxins like poisonous mushroom to 100 oC or higher temperature, will I able to eat it? (I know ...
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1answer
64 views

How long ago did protein synthesis from DNA start? [closed]

How long ago did DNA start to include sequences that code for protein?
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2answers
2k views

Gene vs. Protein Expression Assays

Are the terms gene expression assays and protein expression assays used interchangeably in molecular biology? Or is it expected of you to differentiate between the two terms. For example, if I ...
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1answer
146 views

Reason behind using inducible promoter for heterologous expression

Protocols often seem to use regulatable promoters when expressing a heterologous gene in a host like yeast where they add something like IPTG to induce expression from (say) the lac promoter. Is ...
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1answer
271 views

How to put a gene under the control of a regulable promoter?

Suppose I have a hetrologous (eukaryotic, plant) gene sequence that I want to express in E Coli under the control of a regulable promoter (say the lac promoter induced by IPTG) how exactly does one ...
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3answers
1k views

T7 vs pBAD promoter strength

How do pBAD and T7 promoter strengths compare when pBAD is induced under conditions which would lead to its highest strength? Are there any papers that compare the two inducible promoter systems?
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1answer
35 views

Methods for varying insert size in E. coli clones

I'm writing my master's thesis proposal, and I need to improve the solution-behavior of some truncated protein domains which have previously shown poor solubility when expressed in E. coli. To ...
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0answers
45 views

Computer Models of E Coli / Saccharomyces cerevisiae metabolite pathways

Are there mathematical models one can use to predict the expression levels of a foreign gene inserted into a well studied host organism like E Coli or S cerevisiae? I'm not, obviously, looking for ...
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0answers
275 views

Relative densitometry from SDS PAGE

I'd like to perform densitometry on a Coomassie stained SDS PAGE gel to compare a recombinant protein's expression levels under two conditions. I'm using BioRad's Image Lab software. My questions are ...
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2answers
1k views

What is the best way to express two proteins in a mammalian cell?

I have two proteins and I will be preparing a vector with both genes for stable transfection. Each protein will have their own promoter and I will use piggyBac vector to insert a single cassette with ...
2
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2answers
147 views

Are multi-chain proteins synthesized as one biological unit?

Are multi-chain proteins (especially homo/hetero-dimers) synthesized together as one overall unit or are they separate monomers which bind together at some point after synthesis, and are there any ...
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1answer
2k views

How is the subunit molecular weight different from the native molecular weight?

I noticed that the native molecular weight for an enzyme is different from its subunit molecular weight. Why are they different? Aren't the genes needed to express the enzyme the same in the native ...
2
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1answer
114 views

Do I have to use sucrose to induce a lac promoter?

I'd like to optimize the expression of a Fab fragment in Escherichia coli. For induction of the lac promoter on the pAK400 vector I use IPTG and sucrose. Do I optimize the expression in case I would ...
4
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1answer
167 views

Is it possible to express the cistrons from a polycistronic insertion fragment in a single plasmid?

I have a insertion fragment that I wish to express from pUC19 in Escherichia coli. The insertion fragment is a sub-section from a larger operon sequence and contains just the last two cistrons from ...
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0answers
149 views

Why HIS tag is not detected in WB or dot blot?

I would like to know why the recombinant protein with his tag at c terminus is not being detected neither in western blot or dot blot. I'm trying to validate protein expression of four proteins. ...
2
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0answers
120 views

Which method would more accurately help to identify the unknown concentration of a protein sample between the A280 and the Bradford methods?

I quantified my protein using the standard Bradford method and the A280 methods and obtained values that were far off from the theoretical value of the protein of interest, and therefore was wondering ...
3
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1answer
602 views

Western blot alternatives

Given an in vivo sample from an experimental infection, I would like to see if a bacterial protein is present. I've been thinking about Western Blot using sera from animals infected with the bacteria....
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1answer
46 views

If gene differs between chromosome pair does individual get a mixture of protein shapes?

Do diploids such as humans end up with a mixture of two different shaped proteins where the appropriate gene is expressed differently in the two halves of a chromosome pair or is there some governing ...
5
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1answer
70 views

What's the duration between the binding of adrenaline to β-AR and the first translated protein?

Binding of adrenaline (epinephrine) to the β-Adrenergic receptor leads to formation of cAMP (via G protein activation), activation of protein kinase A and subsequently to the expression of specific ...
5
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1answer
37 views

Need to make a library of mammalian coding sequences

I want to clone ~100 different coding sequences from human or mouse into an expression vector to analyze phenotypes in tissue culture. I can do all of the steps, but I'm not sure what I should use as ...
2
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1answer
184 views

can a bacterial protease inhibitor cocktail be used for Western Blotting involving HUVEC cells and HT-29 adenocarcinoma cells?

Dear fellow biochemists, I need some advice on Western Blotting, more specifically the use of certain protease inhibitors with the RIPA cell lysis buffer and protease inhibitor cocktail. A Millipore ...
5
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3answers
988 views

What are some examples of genes that code for multiple proteins?

The title pretty much says it all. It is widely taught that a gene in a eukaryotic system could produce more than one protein due to post-transcriptional modification, but I do not believe I have come ...
2
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1answer
77 views

Epistasis when interacting loci are codons within a single gene

There is epistasis when the effect on the phenotype of one gene is influenced by one or more other genes (called modifiers). Is there a similar concept when the effect on the phenotype of one site ...
3
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1answer
590 views

What is the translation termination efficiency in mammalian cells?

When I express proteins in bacteria I put at least two stop codons at the end of the gene to increase the termination efficiency. Is this the case in eukaryotic cells too? If I put a single stop codon ...
3
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1answer
4k views

Where to put the gene after eukaryotic promoter for best expression levels?

As far as I know there is an optimum distance between a promoter and the gene for the best expression levels. What is that distance for common promoters like CMV, SV40? If you have a first hand ...
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1answer
90 views

bioinformatics /genomics [closed]

i am looking for likely truncation sites to prove recombination in sequences. i have 4 sequences. i believe one to be the "real sequence" and the other 3 to be recombinants caused by cleavage possibly ...