I would try to use a simpler method to study the dislay levels, which can be very variable from one protein to another and even between variants within the same library If you coat ELISA plates with the anti-flag and detect bound phages with anti-M13/HRP you can titrate the presence of displayed proteins in each phage prep. For intance you could do ten-fold serial dilutions. You could distinguish in that way between no display or low at all and low display levels. This is also a very simple assay to compare different preps from the same or different libraries. It doesnt tell you anything aobut the size of the dislpayed proteins, but if you combine with other experiments you can get valuable information.

For instance, I assume that you checked a sample of your library clones by sequencing and you know that many of them actually contain the correct insert. Is that the case? If not, you could have mostly empty vector in your library and you wont be able to get any displayed protein.

Would be good to check back your library design to see if you inserted any unwanted stop codons. This is assuming that things are fine with your prolongation protocol. You could also sequence one clone with the correct sequence and package it. Jst to confirm your protocol is fine.

Hi Dr. Lawrie,

Make sure your library clones contain the correct insert by sequencing. If not, your library often has an empty vector. You cannot see the target protein. As the insert is located in the target area, you can be sure by designing primers covering the connection points. Also, if a STOP codon occurs in that area after insertion, you may not be able to see the new products. In this case, you can check your library again.

Thank you all for your quick responses. Your insights are very helpful.

Dr. Rojas,

I think your ELISA experiment would be fruitful to try to determine the expression level of the pIII fusion with my protein. I will give it a try as soon as I can and get back to you all with my results.

Dr. Rojas, Dr. Lim and Dr. Duran,

I have sequenced my constructed library several times now with no apparent mistake in the sequence of both my inserted protein of insert DNA sequence or the pIII protein downstream DNA sequence. I used overlapping PCR and primers containing NNK codons to randomized four amino acid positions in my protein so I do expect some to contain stop codons. In saying this, XL1-Blue E. coli the cells I use for phage propagation are capable of suppressing the amber codon with glutamine so only two stop codons are of concern. (the stop codon for the pIII fusion is TAA)

As to more information, the phage derived from a previously developed library using the same protein as my library in the second lane of my Western have already been published and been shown to have Anti-FLAG signal at 39.9 kDa (not 22 kDa as in my results). This is why I am concerned that it may be something in my electroporation and initial infection protocol that may be preventing adequate expression of pIII fusion proteins.

Any more insight would be greatly appreciated. Thank you all.

If you are sure about the library (at the genetic level) then it would be good to produce phage particles with a known insert (for instance, with a phagemid containing the wt gene), to confirm whether the phage production protocol is working properly or not.

I have seen sometimes a dramatic decrease in display after introducing NNK codons, perhaps due to the appearance of multiple variants that do not fold properly.

Something you could do (if sequencing capacity is not limiting) is infecting bacteria with your phages and sequence the inserts to see whether truncated versions of your gene are emerging during phage amplification. It happens sometimes as well.