Herein, we have described a method used for display of random peptides on the surface of bacteriophage in a way that its frequency of display can be regulated. It can be used in multiple applications like characterization of proteolytic activity as done in this case. Other than characterizing substrate specificity of proteases, the technique can be utilized for various other purposes like identifying specific antibodies, interaction with specific ligands and many more. Many publications have come up since the publication by Cwirla et al., (1990) for identifying ligands displayed on the surface of a bacteriophage. In fact, there have been so many biotech companies as well, working in the pursuit of identifying suitable substrates as well as inhibitors using this technique. We have used this technique for identifying specific substrates for some proteolytic enzymes like implantation serine proteinase (a native dimer), recombinant ISP1, recombinant ISP2 along with human kallikrein 6 (KLK6). The protocol used has been described here in detail. [1] [2] [3] [4] [5] [6]

The following materials are utilized for the construction of T7 phage-displayed peptide library:

1. T7 select 1-1 vector arms
2. T7 select system (M/S Novagen Canada: EMD Biosciences Inc., an affiliate of Merck KGaA, Darmstadt, Germany)
3. Trypsin (M/S Sigma)
4. E. coli (BLT 5403) – obtained from ATCC
5. Magnesium Sulfate (M/S sigma)
6. Agarose (M/S HiMedia)
7. NiNTA Agarose Beads (M/S GE)
8. Tris.Cl (M/S Sigma)
9. PCR (M/S BioRad)
10. PCR Purification Kit (QIAquick from M/S Qiagen)

Construction of the phage display library

1. Start by constructing the oligonucleotides that need to be displayed on the surface of bacteriophage. The sequence of synthetic oligonucleotides inserted in the coding region of the T7 phage capsid (employing T7 select 1-1 vector arms, T7 select system, Novagen Canada; EMD Biosciences Inc., an affiliate of Merck KGaA, Darmstadt, Germany) is shown here:
- 5'-AAT TCT CTC ACT CCA GGC GGC-(NNK)₆GGT GGT CAT CAC CAT CAC CAT CAC TAA-3' whereas N represents any nucleotide and K represents thymine (T) or guanine (G)
- CTC, ACT and CCA are the nucleotide codes for leucine, threonine and proline. It is part of the linker and helps in isolation of the random peptide.
- (NNK)₆ represents the oligonucleotides for random hexamer of amino acids. N represents any nucleotide and K represents thymine (T) or guanine (G).
- GGC GGC and GGT GGT are the nucleotides that represent flexible glycine dimers at either end.
- CAT CAC CAT CAC CAT CAC are nucleotides that represent a (His)6 tag at the C-terminus.
- TAA represent a STOP codon at the 3'-terminus of oligonucleotide.
2. Three oligonucleotides are constructed as given below:
A. AAT TCT CTC ACT CCA GGC GGC-(NNK)₆-GGT GGT CAT CAC CAT CAC CAT CAC TAA
B. GCC GCC TGG AGT GAG AG
C. A GCT TTA GTG ATG GTG ATG GTG ATG ACC ACC
Different concentrations of these oligonucleotides are made up for use in the construction of the library. 2 µM of A, 40 µM of B and 40 µM of C is made up in the following buffer:
10 mM Tris.Cl (pH 8.0) and 0.2 mM EDTA
3. T7 phage DNA vector (Novagen, Canada) digested by EcoR I and Hind III is used for ligation of the oligonucleotides. The ratio for ligation between T7 phage vector and oligonucleotides A, B and C is 1:5:100:100, i.e., 0.004 µM of vector, 0.02 µM of oligonucleotide A and 0.4 µM each of oligonucleotide B and C. 5X oligonucleotide concentration will be used for annealing for 1 µl of annealed oligonucleotides in 5 µL ligase reaction. That means 0.1 µM of oligonucleotide A and 2 µM each of oligonucleotide B and C.
4. The annealing is done in 100 µL volume in the ratio as given below:
5X oligonucleotide annealing buffer -20 µL
2 µM oligonucleotide A -5 µL
40 µM oligonucleotide B -5 µL
40 µM oligonucleotide C -5 µL
DDW -65 µL
Heat it to 70oC for 5 minutes and let it cool slowly to room temperature.
5. Ligation is done in 5 µl total volume. But before ligation, the de-phosphorylated vector is re-phosphorylated once again as given below:
T7 DNA vector (EcoR I-Hind III digested
-1.0 µL
and de-phosphorylated)
10X Ligase buffer (containing ATP, DTT -0.5 µL
but no PEG)
DDW -1.0 µL
T4 polynucleotide kinase -0.5 µL
Annealed oligonucleotides -1.0 µL
Incubate it at 37°C for 20 minutes. Add 1.0 µL T4 DNA ligase and incubate at 16°C overnight. Store at 4°C until in vitro packaging.
6. Thaw the in vitro packaging extract (T7, Novagen, Canada) on ice in the ice bucket. Do not freeze thaw quickly at all. Add ligation reaction (5 µl) to this extract. Mix gently maybe with a pipette-man but do not froth. Stop the reaction by adding 270 µL LB after incubating it for two hours at room temperature.

Titration

1. Inoculate BLT5403 strain of E. coli in M9TB medium at 37°C. Let OD₆₀₀ reach 1.0 and store at 4°C (up to 48 hours).
2. Prepare molten top agarose in a bottle and keep it at ~50°C.
3. Prepare a series of dilutions using LB as the diluent in 1.5 mL eppendorf tubes. Make the following dilutions as given below:
10^-2 dilution - 10 µL Phage + 990 µL LB
10^-3 dilution - 100 µL phage from 10^-2 dilution + 900 µL LB
10^-4 dilution - 100 µL phage from 10^-3 dilution + 900 µL LB
10^-5 dilution - 100 µL phage from 10^-4 dilution + 900 µL LB
10^-6 dilution - 100 µL phage from 10^-5 dilution + 900 µL LB

4. Mix 3 mL top agarose with 100 µL dilution as shown above and 250 µL BLT5403 host cells. Pour it over a pre-warmed LB-Agar (Amp+) plate.
5. Incubate for three hours at 37oC, or overnight at room temperature.
6. Count the number of plaques per plate and estimate the total number of phages in the library.

Amplification of the library

1. Grow BLT5403 culture to 500 mL in LB medium (+ 50 µg/ml ampicillin) at 37°C. Grow until OD600 = 0.5–1.0.
2. Infect the bacterial cell culture with unamplified phage library. Add all of the phage particles including the ones in the remaining 10-2 dilutions made for estimating the titre.
3. Incubate the culture at 37°C with shaking for 1–3 hours until lysis is observed.
4. Spin the culture at 8000 x g in JA-10 rotor (6730 rpm) for 15 minutes. Decant the supernatant back into sterile bottles.
5. Store the amplified phage library at 4oC.
6. Count the number of plaques/mL in the library as per the titration method given in section titration. Since the number of plaques is high in the amplified library it is better to look for the number in 10^-6 – 10^-10 dilutions.

PCR Analysis Plaques were eluted from a plate having less than 100 colonies so that it is not difficult to pick the plaques. Single plaques were eluted from the plate into 200 µL SM (Recipe given in the media recipes). Do the PCR in 25 µL reaction volume, without pre-heating and without hot start. Here is the recipe for PCR reaction:

Plaque lysate -1.0 µL
DDW -19.075 µL
50 mM MgCl2 -0.80 µL
10X PCR Buffer (no Mg2+) -2.5 µL
5 µM T7 Select UP Primer -0.5 µL
5 µM T7 Select DOWN Primer -0.5 µL
10 mM each dNTPs -0.5 µL
Taq DNA polymerase -0.125 µL
Follow the following temperature conditions at different steps: a) 94oC - 3 min b) [94oC - 0.5 min; 52oC - 0.5 min; 72oC - 1 min] x 38 cycles c) 72oC - 6 min Incubate at 4oC till further use.

Biopanning Subsequent to binding of amplified phage displayed library (1x1010 pfu) with 100 µL of Ni-NTA agarose beads (Sharma et al. , 2008); biopanning is performed with 10 µL of trypsin (Sigma). A control elution is performed using 500 mM imidazole solution. Repeat the process five times and obtain the plaques after lysis of the E. coli (BLT5403) culture upon infection with plaques. These plaques are plated on LB agarose plates as explained in the titration section above. The plaques from these plates can be amplified and picked for amplification of DNA by PCR using T7 primers. The PCR product was purified using QIAquick PCR purification kit (Qiagen, Canada). These DNA samples are sent for sequencing after PCR and the amino acid sequences are deduced from there.

Characterization The results of sequencing reactions give the amino acid stretches in each reaction as a result of the DNA sequence of each plaque. The results are compiled in a tabular form and then seen if they represent any uniformity or not. As is evident from the results, the substrate specificity results of trypsin are similar to its published data.
The sequencing reactions results after PCR of the library are given in Table 1. The amino acid content of each amino acid in the above sequenced reactions before biopanning is given in Table 2. Figure 1 describes the different steps in the process of using phage display library for substrate specificity determination.

It is clear from the results that most of the amino acids show random display in the library. The results could be improved by sequencing a greater number of plaques. The results obtained after biopanning with trypsin are exactly as expected.

AA: Amino Acid, % Obs: % Observed, % Nom: % Nominal, G: Glycine, R: Arginine, V: Valine, T: Threonine, Y: Tyrosine, S: Serine, L: Leucine, E: Glutamic Acid, P: Proline, A: Alanine, C: Cysteine, I: Isoleucine, M: Methionine, K: Lysine, F: Phenylalanine, Q: Glutamine

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2. Sharma N, Kumar R, Renaux B, et al. Implantation serine proteinase 1 exhibits mixed substrate specificity that silences signaling via proteinase-activated receptors. PLoS One 2011;6(11):e27888.   [CrossRef]   [Pubmed]    
3. Sharma N, Oikonomopoulou K, Ito K, et al. Substrate specificity determination of mouse implantation serine proteinase and human kallikrein-related peptidase 6 by phage display. Biol Chem 2008 Aug;389(8):1097–105.   [CrossRef]   [Pubmed]    
4. Karlson U, Pejler G, Froman G, Hellman L. Rat mast cell protease 4 is a beta-chymase with unusually stringent substrate recognition profile. J Biol Chem 2002 May 24;277(21):18579–85.   [CrossRef]   [Pubmed]    
5. Deperthes D. Phage display substrate: A blind method for determining protease specificity. Biol Chem 2002 Jul-Aug;383(7-8):1107–12.   [CrossRef]   [Pubmed]    
6. Cwirla SE, Peters EA, Barrett RW, Dower WJ. Peptides on phage: A vast library of peptides for identifying ligands. Proc Natl Acad Sci U S A 1990 Aug;87(16):6378–2.   [CrossRef]   [Pubmed]