Generaly with E.coli expression you are trying to slow down the rate of transcription & translation to enable the production of of correctly folded soluble protein. This is more important than total expression levels. Having a low copy number plasmid is just another part of this whole.

I have however never done any exhaustive comparison between high and low copy plasmids, and in most cases there would be other differences between the plasmids that could also affect the expression levels.

In reality it is the protein that is driving the expression levels, and hence the choices, and the results, rather than the vector copy number. So the system that gives you what you want is the best system, rather than some generalisation that this or that is the 'best'.

-Richard

It all depends on the kind of protein you are going to express I guess. If the protein is toxic, it is better to use a low copy plasmid to get some protein (high copy no will kill your host).

http://bitesizebio.com/22824/how-to-manipulate-plasmid-copy-number/

Hi there,

The plasmid copy number should be considered in combination with the strength of the promoter. For instance, for T7 system (Studier), low copy plasmid is enough because the level of transcription is high.

what I think is:

1.High copy number will lead to more protein and use of the cell energy towards protein formation so the other processes will get disturbed due to lack of energy. so this is toxic to cell.

2.further the high copy number plasmid increase protein will not have proper folding and formation of inclusion bodies occur accumulation these bodies will lead to problem.

3. compare to this low copy number plasmid lead to low protein production and properly folded protein formation thats why they recommend.

now a days lots of commertial plasmids are available people use them.

Generaly with E.coli expression you are trying to slow down the rate of transcription & translation to enable the production of of correctly folded soluble protein. This is more important than total expression levels. Having a low copy number plasmid is just another part of this whole.

I have however never done any exhaustive comparison between high and low copy plasmids, and in most cases there would be other differences between the plasmids that could also affect the expression levels.

In reality it is the protein that is driving the expression levels, and hence the choices, and the results, rather than the vector copy number. So the system that gives you what you want is the best system, rather than some generalisation that this or that is the 'best'.

-Richard

High Copy expression vector may reach ~300 copy per E. coli in stationary phase which is about 15 folds of low copy vector. So that the high copy vector is prone to leakage expression due to higher lacI to lacO ratio. When expressing a toxic protein, the leakage expression in the absence of inducer, IPTG, will lead to selection of low expression clone. In the other hand, the low copy expression vector provides better expression regulation that keep protein expression in check. Other option when encounter toxic protein is to use competent cell that contains pLysS, a T7 RNA polymerase inhibitor, that gives even tighter expression regulation. 

Dear Arthur,

Your question is a wonderful one. As other answers, promoting strong transcription and translation could be deleterious if your protein is overactive, if your gene uses rare codons and if your protein is not folding right. I suggest you to use a high copy vector and control the transcriptional expression of your gene, which should contain the best Shine-Dalgarno as possible. Then test the expression with a gradient of your inducer molecule to check for level of expression, toxicity and folding (regularly by checking for protein precipitation or cellular stability). Low copy plasmids are fine, but they usually are for expressing genes as close as chromosomal levels to study cellular activity.

High copy number plasmid results in increased copy of a specific gene per cell, and invariably higher amount of a specific protein synthesized per cell, compared to low copy number plasmid. Now, as the process of transcription doesn't involve proof reading, the higher the copy of a gene transcribed, the higher the rate of transcription error. This, coupled with the number of protein translation per gene increases the chances of improper protein folding, and thus higher chance of inclusion body formation. This is particularly the case for constitutively expressed genes. For inducible system where transcription of particular  gene is regulated and repressed, the situation is a bit different. Here, the higher number of plasmid copy per cell is commensurate with high number of operator per cells. However, as the repressor is constitutively expressed, the aforementioned negative effect of high copy number on expression of correctly folded protein negatively affects the number of repressor molecules thus lowering the repressor/operator ratio in the cell. This leads to leaky expression of the regulated protein (as there will be plasmid copies where the operator is unbound to a repressor). Generally leaky expression is bad especially when the regulated protein is toxic to the cell, or in cases where controls are required. That said, a moderately medium copy number plasmid may be better than a low copy number plasmid as the former allow for a wider range of induction and more protein expression with lower chances of improper foldiing.

I use high copy vectors only for cloning purposes to increase the number of gene copies, for example, when I make Widom601 DNA fragments for nucleosome refolding, or when I use GIbson assembly. From my experience the copy number does not affect your protein expression at all (even for toxic proteins), because it is likely not a rate determining step when strong promotors are utilized. 20-50 copies per cell is sufficient to sustain high level of transcription. In my experience promoter type matters a lot - with some proteins I get way higher expression with phoA (Pi induction) and rhaBad (rhamnose induction, KRX E.coli cells ) promotors compared to T7 (IPTG or lactose) in vectors of comparable size, antibiotic resistance, and with the same replication origin. I just recalled that there was only one time when the high copy plasmid we received from a collaborator gave us horrible result compared to low copy - it was a strong DNA binding protein that was apparently aggregating in the presence of excessive plasmid copy number (200 per cell).

Using low copy number plasmids by default is ideal. Transcription-replication conflicts are minimised in low copy number plasmids. This aids in the production of non-truncated, thus correctly folded soluble protein. Unresolved conflicts are expected because plasmids are exogenous irrespective of copy number; But, transcription-replication conflicts are resolved to the greater extent in low copy number plasmids than high copy number (Because of titration of conflict resolving complex proteins). However, depending on the type of system like mammalian cell model, high copy number plasmids are often used for expression.