Phage Display Peptide Library-Special Topic on Phage Display Technology-Tekbiotech-Yeast Display Service,Phage display technology

Phage Display Peptide Library

I. What is Phage Display Technology?

Phage display describes an in vitro selection technique in which a library of variants of a peptide or protein is expressed on the outside of a phage virus, while the genetic material encoding each variant is inside. This creates a physical link between each variant protein sequence and the DNA encoding it, allowing the variants to be screened based on their binding affinity to a given target molecule (antibody, enzyme, cell surface receptor, etc.). The simplest screening method is to incubate the phage-displayed peptide library with a plate (or beads) coated with the target, wash away unbound phage, and elute the specifically bound phage. The eluted phages are then amplified and the binding sequences in the pool are repeatedly enriched through more binding/amplification cycles. After three or four rounds, individual clones are identified by DNA sequencing and ELISA. The information collected in such an experiment may be an end point in itself, i.e., determining the epitope sequence of a monoclonal antibody, or it may be just the beginning of the search for a viable agent, since peptides are generally neither stable nor biologically active in vivo.

II. Filamentous Phages

Filamentous phages (also commonly called phages) are a class of viruses that infect Gram-negative bacteria containing F plasmids, such as Escherichia coli cells, some of which are classified in the Inovirus family and belong to the Inovirus genus, such as phages M13, f1, and fd. Phages can be considered as cloning vectors and require helper phages to provide the structural and functional proteins required to package the phage into virions to complete their infection process.

Infection begins with the attachment of the N-terminus of pill to the tip of the F-pilus on the bacterium. This binding results in the injection of the ss-DNA (+) strand of the phage into the bacterial cell. The host polymerase then uses the (+) strand as a template to produce the complementary strand (-), thereby forming the double-stranded (ds-) or replicative form (RF) of the phage. Phage proteins are synthesized from mRNA generated from the RF DNA strand (+). To replicate the genome to produce new phages, the newly synthesized protein pII cleaves the RF DNA to initiate the replication of the (+) strand. As a result, the host enzymes can produce a large number of RF DNA molecules. pII also connects the molecular ends of the newly synthesized (+) chain to form ss-DNA. The pV protein dimer binds this new ss-DNA to prevent conversion to RF DNA. The amount of pV determines the ratio of RF to (+) chain DNA synthesis. pX is involved in replication and is thought to regulate RF/(+) chain DNA synthesis as well as inhibit pII function.

Assembly occurs at the inner membrane of the cell and includes pI, pIV, and pXI. The C-termini of pI and pXI interact with pIV to form a channel that promotes the secretion of the phage. pVII and pIX are required for the secretion step and interact with the pV-ss-DNA complex. During extrusion, pV bound to ss-DNA is replaced by pVIII, and then pVI and pIII are added at the proximal end of the released particle.

Structure of bacteriophage and its life cycle-tekbiotech.png

Figure 1 Structure of bacteriophage and its life cycle

III. Peptide Library Construction

Each of the 20 natural amino acids is encoded by a codon. Using degenerate oligonucleotides introduced into the phage genome, random peptide libraries can be constructed. One of the most commonly used strategies for generating random peptides is to use (NNK), a codon degenerate system where N is the four nucleotides (adenine, guanine, cytosine, and thymine) and K is a 1:1 equimolar mixture of guanine and thymine. By using (NNK)n codons instead of (NNN)n codons, the number of stop codons is reduced from three types (TAA, TGA, and TAG) to one type (TAG, Amber stop codon). For a peptide library using (NNK)n codons, each NNK is a mixture of 32 different possible codon sequences that encode all 20 amino acids (plus one stop codon). For such a library, the number of possible n-mer peptide sequences is given by 20n, where 20 is the number of standard amino acids and n is the number of randomized positions. For example, for a peptide library with 7 random positions, there are 20^7 (1.3×10^9) possible heptamers. However, these considerations are an oversimplification of reality and can lead to overestimations for a number of reasons, including the degeneracy of the amino acid code leading to the random occurrence of stop codons and transformation efficiency. The maximum concentration of phage particles is 10^14 particles/mL (170 nM), which sets an upper limit on diversity. Transformation efficiency is limited to 10^8-10^10 phage constructs transformed into E. coli by electroporation or other techniques.

Peptide libraries can generate displayed peptides ranging in length from 6 to 30 residues. Strategies can be used to present peptides in more constrained conformations; for example, by including two cysteine residues to form a disulfide bond. It is difficult to predict the optimal length required for randomly displayed peptides because it depends on many factors, including the folding properties of the displayed peptide, the properties of the target, and the purpose of the study.

Based on the phage display platform, TekBiotech can construct random 7-peptide libraries, 12-peptide libraries, cyclic 7-peptide libraries, etc. It can not only provide customers with a variety of linear peptide library construction services based on T7 phage and M13 phage, but also provide customers with high-quality cyclic peptide phage display library construction and screening services.

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