Guys,
I want to understand more about the function about helper phage and I start reading. One articles says that 'When wild-type phages are used, interference of the plasmid with the phage replication leads to reduction in the phage copy number and drastic decrease in viron production'.
Could anyone help explain this sentence? Why plasmid can interfere with viral particle production?
There are several things to be corrected in the above statements.
1. The phagemid is responsable for the expression of scFv-PIII, not the other phage proteins. No genes coding for the viral proteins (except scFv-PIII) are found in the phagemid. No viral particles at all are produced if you have phagenid-transformed cells.
2. The other viral proteins come exclusively from the helper phage. If you infect cells with the helper phage, new viral particles (helper phage) are produced.
3. If you infect with helper phage phagemid-transformed cells, new viral particles are produced. They contain all the viral proteins (coming from the helper phage), a low proportion of scFV-PIII (coming from the phagemid) and as phagemid is more efficiently packaged tan the helper phage DNA, most of them contain phagemid inside. A minor fraction would contain helper DNA instead.
4. Regarding the expression of scFv-PIII, usually most viral particles do not have the foreign protein, just wild-type PIII coming from the phagemid. 1-10% of the viral particles display the foreign protein (scFv), normally in a monovalent way. This low abundance of the foreign protein is a way to achieve monovalency. If you prefer multivalent display, it would be better to use modified helper phages or tu display the foreign protein fused to PVIII.
Can you point us exactly to the article in question or provide a bit more context? The statement above is indeed hard to understand.
Helper phages are used for packaging phagemids into viral particles. Helper phages such as M13k07 carry a modified f1 ori region that is recognized less efficiently by their own packaging machinery than the wild type f1 ori borne by phagemids. This way, when you use a helper phage to infect a cell that has been previously transformed with a phagemid, the yield of packaged phagemids is much higher than the yield of packaged helper phage.
Using a wild-type phage would simply lead to much higher titers of packaged phage, and much lower titers of packaged phagemid.
Due to the reasons described above, M13KO7 is commonly used as a suitable helper phage for display based on phagemids...
The sentence is anyway difficult to understand.
Thank you Alejandro adn Gertrudis
it is from this website: http://www.abdesignlabs.com/technical-resources/helper-phages/
It is in the first paragraph!
Thank you!
I believe Alejandro pretty much explained the statement you wrote - when screening phagemid-based phage display libraries, wild type phages, if used as helper phages, can easily overgrow the phagemid-packed viral particles due to the rapid amplification of the first.
In addition to the modified f1 ori some helper phages may have a disrupted packaging signal seguence, so they are packed less efficiently into the emerging phage particles during phage amplification step.
Then you also may have some other modifications in helper phages such as deleted (knocked-out) gene III for polyvalent display of scFvs (or other proteins) like in helper phages R408d3, VCSM13d3 (http://www.ncbi.nlm.nih.gov/pubmed/9370269) or M13cp-CT (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1693883/).
Also, helper phage KM13 may be very useful for screening scFv libraries as it contains a trypsin cleavage site within the p3 which allows for trypsin-mediated inactivation of residual helper phages before amplification steps between biopanning cycles.
Good luck!
Thank you Peter, Gerturdis and Alejandro.
Let me summarize and see whether there is any mistake, please correct me:
When amplifying the phage, we actually use two plasmids to do two different jobs:
(1). The phagemid with pIII-scFv fusion protein gene is responsible for expression the pIII-scFv proteins and also other constituent viral proteins. Phagemid has to be replicating for future packaging. However, because of the speed of protein expression from the phagemid is much slower than the phagemid replication, the packaging is less effective and viral particles is produced much inefficiently.
(2). Helper phage comes to help to boost the production of viral constituent proteins. At the same time, the mutated packaging signal in F1 orgin provides less efficient helper phage self-packaging. Phagemid, instead,now with abundant viral proteins is also in a faster way to be packaged into the phage.
(3) The result is that: In your final phage solution, after phage is collected, you can get the following phage: [1] Monovalent or polyvalent scFv with phagemid, [2] Monovalent or polyvalent scFv with helper phagemid(much less), [3] phage with either plasmid (ssDNA) but without scFv.
Please correct me if I am wrong.
Thank you again.
There are several things to be corrected in the above statements.
1. The phagemid is responsable for the expression of scFv-PIII, not the other phage proteins. No genes coding for the viral proteins (except scFv-PIII) are found in the phagemid. No viral particles at all are produced if you have phagenid-transformed cells.
2. The other viral proteins come exclusively from the helper phage. If you infect cells with the helper phage, new viral particles (helper phage) are produced.
3. If you infect with helper phage phagemid-transformed cells, new viral particles are produced. They contain all the viral proteins (coming from the helper phage), a low proportion of scFV-PIII (coming from the phagemid) and as phagemid is more efficiently packaged tan the helper phage DNA, most of them contain phagemid inside. A minor fraction would contain helper DNA instead.
4. Regarding the expression of scFv-PIII, usually most viral particles do not have the foreign protein, just wild-type PIII coming from the phagemid. 1-10% of the viral particles display the foreign protein (scFv), normally in a monovalent way. This low abundance of the foreign protein is a way to achieve monovalency. If you prefer multivalent display, it would be better to use modified helper phages or tu display the foreign protein fused to PVIII.
Could anyone adivse me on helper phage for polyvalent phage display? I need detailed information.
Look at papers related to hyperphage. This helper phage does not contain any PIII gene, so all PIII copies in that system are fused to the foreign protein.
A different alternative is to fuse your protein to PVIII without changing helper phage. As there are thousands of copies of PVIII, display of your protein on some of them results in polyvalent display.
Oh yes, but I wanted to know how to produce the pIII-deficient helper phages, whereas this protein is necessary for the infection of cells transformed with phagemid. Sorry for my English
I think they are produced in a modified strain able to produce PIII. So they do have PIII outside, but not the coding gene inside. Have a look on papers by Stefan Dubel...
This is a late answer, but most of the above explanations are incorrect. Phage need certain number of copies of double stranded DNA to produce enough viral proteins for phage assembly. When a high copy number plasmid containing the same f1 origin is present in the same cell, due to the ratios of available origins, gene 2 will mostly amplify the plasmid, leaving not enough phage DNA for protein production, therefore greatly reduced viron production, hence the interference. M13KO7 contain another p15A origin to produce enough DNA for phage protein production, therefore it's interference resistant.
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