In recent years, phage display technology has developed rapidly, and it has a wide range of applications in the research fields of antibody epitopes, protein inhibitors and activators, enzyme-substrate specificity, protein folding, drug and vaccine design, gene expression regulation, and molecular recognition in the immune system. The so-called phage display is the method of molecular cloning to express the foreign nucleic acid fragment in the form of fusion protein on the surface of phage particles. In general, phage display experiments can be summarized in two steps: a to build a library; b screening the library, firstly through artificial synthesis, cDNA method, DNase I random hydrolysis method to prepare a variety of nucleic acid fragments with different sequences, and then clone them in the carrier (bacteria or bacteria granules), and then infect or superinfect Escherichia coli to secrete phages with fusion expression of foreign fragments, collectively called library construction. Then immobilized target molecules were used to screen phages that were compatible with them. Phage display technology has two obvious advantages. One is that phage is easy to expand, and the other is that the amino acid sequence of foreign peptides or proteins expressed by fusion can be deduced by measuring the sequence of phage DNA. Phage display can be fused to express peptides, protein domains, and proteins, and the fused peptides displayed on the bacteria are called phage peptides.

This article will discuss the important position of phage display of fusion peptide expression. The phage display library can be divided into two categories according to whether the conformation of the exogenous fused polypeptide is restricted. One is the conformation-restricted peptide library, which uses a pair of disulfide bonds to cyclize the exogenous fused polypeptide. The other class is a conformationally unrestricted peptide library. That is, no disulfide bond binds the conformation of the exogenous fused polypeptide. Whether the exogenous fused polypeptide is expressed on the phage outer membrane in single-copy or multi-copy form, it can be divided into monovalent polypeptide phage display and polyvalent polypeptide phage display accordingly. The following two representative examples will be presented to introduce and evaluate the display of polypeptide phage infiltrating various biological research fields. First, a polypeptide phage display library for screening polypeptide ligands bound to various biomolecules will be outlined, called the ligand phage display library. Subsequently, the substrate phage display library will be introduced to study the substrate specificity of enzymes.

 

1. Ligand Phage Display Library

 

More and more immobilized target molecules are used to screen for ligand phage display library, which expands the application range of bacteriophage display technology. The following will outline the ligand phage display library for screening polypeptide ligands according to the different immobilized target molecules used in the screen library.

 

1.1 Antibodies

 

Scott et al. constructed FUSE5 bacteriophage as a carrier to express random hexapeptides and inserted two amino acid sequences of DGA and GAAGA on both sides of the random hexapeptides, respectively, to minimize the influence of phage outer membrane protein on the conformation of hexapeptides fused in the n-terminal. Scott et al. used two monoclonal antibodies, M33 and A2 (both specifically bind to the antigenic determinant of earthworm myoglobin: DFLEKL), to screen the random hexapeptides. Most of the hexapeptides screened were similar to the antigenic determinant DFLEKL, with the first three amino acid sequences being DFL and the last three amino acid sequences being different. However they also sifted out a hexapeptide CRFVWC with little resemblance to the antigenic determinant DFLEKL, which ELISA showed to bind strongly to M33, an amino acid sequence with little resemblance to the original antigenic determinant (in this case, DFLEKL) called Mimotope. This experiment shows that the antigenic determinant of monoclonal antibody may be known by screening random peptide libraries without knowing the antigenic determinant of monoclonal antibody in advance, and sometimes some peptides that are very different from the amino acid sequence of the original antigenic determinant can be screened, which enriches the structural information of antigen-antigen interactions.

 

1.2 Lectins

 

Lectins with ConA (ConA) specifically bind methyl-alpha mannose (MaM). Scott et al. used ConA to screen the random hexapeptides, and the obtained bacterial polypeptides had the characteristic sequence YPY, and the binding of ConA to these bacterial polypeptides could be inhibited by the natural CONA ligand MaM. Binding experiments with other similar D2 mannose-binding lectins showed weak binding of these phage peptides to them, indicating high specificity of these phage peptides in binding ConA. Oldenberg et al. used ConA to screen polypeptides with the characteristic sequence YPY from the random octapeptide library. It can be seen that ConA ligands are not limited to sugars, and it is possible to screen saccharide-mimicking polypeptides from random peptide libraries.

1.3 Major Histocompatibility Complexes

Major histocompatibility complex Class II molecules are highly polymorphic membrane glycoproteins that bind to cleaved peptides of proteins and present them on the surface for recognition by CD4+T cells. Hammer et al. used three related major histocompatibility complexes Class I alleles (HLA2DRB1 0101, 1401, 1101) to screen the random nine-peptide library, and accordingly obtained three motifs YX₂MXAX₂L, WX₂MXTLX₂, and WX₂MXRX₃. X represents any common amino acid (the same as below). The three have two anchor residues similar or identical at positions 1 and 4, showing their specific anchor residues at position 6. Therefore, the phage library of polypeptides is also helpful for studying the interaction between polypeptides and major histocompatibility complexes.

 

1.4 Enzymes

 

Bovine pancreatic ribonuclease was partially hydrolyzed with subtilisins protease to obtain two fragments: S2 peptide (20 amino acids) and S2 protein (104 amino acids). Both have no enzyme activity individually and only show enzyme activity after binding. The S2 peptide and S2 protein system is considered a good model for studying the interaction between receptors and ligands. Smith et al. used S2 protein to screen the random hexapeptide library, and the phage peptides screened had the characteristic sequence (F/Y)NF(E/V)(I/V)(L/V). This characteristic sequence is very different from that of S2 peptide. However, inhibition experiments with YNFEVL, a synthetic polypeptide with this characteristic sequence, showed that YNFEVL competitively inhibits the binding of S2 peptide to S2 protein and is an antagonist of S2 peptide. This experiment has implications for the discovery and study of antagonists, agonists, and drugs.

 

1.5 Nucleic Acids

 

Krook et al. screened random hexapeptide libraries with single-chain 7 polycytidylate in two buffer liquid systems (pH 5.5 and pH 7.5), respectively. Three phage polypeptides (PPPLYF, RFCDTS, RSR2LIW) strongly binding to single-chain 7 polycytidylate were screened in the pH 5.5 buffered system. In recent years, there has been a great increase in interest in DNA-specific peptides. Because these peptides may be used to regulate the expression of specific genes in cells selectively, providing information for the design of antiviral and tumor drugs, it is convenient to use random peptide libraries to study the interaction between nucleic acids and peptides.

 

1.6 Receptors

 

Thrombin receptors on platelets are membrane proteins with seven transmembrane alpha helices, belonging to the G-protein-coupled receptor superfamily, whose six N-terminal amino acids bind to and activate the receptor as tethered ligand after the extracellular N-terminal is digested by thrombin. Doorbar et al. used platelets containing thrombin receptors to screen the random peptide library, from which phage polypeptides (MSRPACPNDKYE) that could immunoprecipitate thrombin receptors were screened. The phage polypeptide was synthesized to antagonize platelet agglutination, tyrosine phosphorylation, and release of 5-hydroxytryptamine by agonists of the receptor. They also screened phage peptides that activate the receptor.

1.7 Calmodulins

Calmodulin plays an important role in intracellular signal transduction pathways by binding to many target proteins or enzymes and regulating their functions. Dedman et al. screened a random pentapeptide library with calcium-dependent calmodulin, and found 28 bacterial polypeptides, all containing a tryptophan, among which W2P appeared very frequently. These synthetic bacterial polypeptides can bind to calcium-binding calmodulin and compete with known calmodulin inhibitors.

1.8 SH Homologous Domain

Many proteins involved in intracellular signal transduction pathways contain a domain of 50 to 60 amino acids, known as the SH3 homology domain. The SH3 homology domain mediates protein-protein interactions in signal transduction pathways. Previous studies have shown that the recognition sequence of the SH3 homology domain is PXXP and Rickles et al. further determined the feature recognition sequence of SH3 homology domain of five proteins (Src, Fyn, LynP13K, and Ab1) by using the polypeptide phage display library. The second is XXXRPLPPLPXP. XXXRPLPP(I/L)PXX, RXXRPLPPLPXP, PXXRPLP2PLPPP, RXXRPLP2(I/V)PXX, This provides valuable information for the study of SH3 homologous domain-mediated signal transduction pathway and drug design targeting this pathway.

1.9 Integrins

Integrin is a class of transmembrane glycoproteins involved in cell adhesion. All known integrins are heterodimers. α₅β₁ integrin recognizes the RGD sequence of fibronectin and binds to it. Koivunen et al. used α₅β₁ integrin to screen 32 different peptide ligands of α₅β₁ integrin from the random hexapeptide library, among which 28 contained RGD motif, 3 contained RGD-related sequences, and one phage multidermic peptide (SLIDIP) was very different from RGD. In particular, the phage polypeptide CRGDCL contained two cysteines in its sequence so that disulfide bonds may limit its conformation, and it can inhibit the binding of cells expressing α₅β₁ integrin to fibronectin with 10 times greater efficiency than other linear hexapeptides containing RGD sequences.

 

Fig 1 Integrin Structure and Activation

1.10 Molecular Chaperones

The chaperone Bip is located in the endoplasmic reticulum of eukaryotic cells and assists in the folding and assembly of newborn peptides through the endoplasmic reticulum and subsequently in the endoplasmic reticulum lumen. The role of Bip depends on its ability to recognize a large number of new peptides with less similar amino acid sequences and its ability to recognize correctly folded and unfolded protein structures. Blond-Elguindi et al. screened BIP-bound phage polypeptides from random octapeptide and dodecapeptide libraries with the characteristic sequence Hy (W/X) HyXHyXHy, which alternates large hydrophobic amino acids (Hy) at odd positions. Hy is usually tryptophan, leucine, or phenylalanine. Thus Blond-Elguindi proposed a model that the polypeptide binding site of Bip is composed of alternating hydrophobic amino acids. Synthetic peptides with this characteristic sequence can bind to Bip and activate its ATPase activity. Therefore, polypeptide phage library can be a powerful tool for studying protein folding.

 

 

Fig Mechanism of action of chaperone BiP

2. Substrate Phage Display Library

 

The substrate phage display library has been successfully used to study the substrate specificity of proteolytic enzymes and protein kinases. The following will introduce the substrate phage display library to study the substrate specificity of these two types of enzymes respectively.

 

2.1 Proteolytic enzymes

 

Matthews et al. used the monovalent phage display library to study the substrate specificity of proteolytic enzymes. They constructed two substrate phage display libraries with intercalated fragments GPGG(X)5GGPG and GPAA (X)5AAPG, respectively, and used them to study the substrate specificity of subtilisin protease and human Factor Xa. According to the sequencing of phage, the hydrolyzed substrate of subtilisin protease contains histidine, which is mostly distributed in the second or fourth position of random polypeptide sequence. Polypeptide sequences resistant to subtilisin protease hydrolysis are characterized by one or more prolines and no histidine. Human Factor Xa substrate sequences are characterized by almost all containing at least one arginine, with four clones containing two arginine.

Matthews et al. also investigated the substrate specificity of furin using a substrate phage display library. Flynn's protease is a mammalian enzyme that cuts many constitutively expressed protein precursors. After six rounds of screening, the recognition sequence of Flynn's protease was deduced to be RXXR, and many of the clones screened were often lysine or arginine, or proline before the second arginine.

 

2.2 Protein Kinase

 

Eukaryotic M-phase (division phase) protein kinases phosphorylate a variety of proteins, and phosphorylation of these proteins may be important for cell division. Westendof et al. used the random pentapeptide library and the monoclonal antibody MPM2, which can recognize and bind to the sequence of phosphorylated amino acid residues in more than 40 proteins of M-phase eukaryotic cells, to study the substrate specificity of M-phase protein kinase. They first dephosphorylated the random pentapeptide library with M-phase protein kinase. Those phage peptides phosphorylated by M-stage protein kinase are screened for binding to monoclonal antibody MPM2. It is deduced by sequencing that the characteristic phosphorylation recognition site of M-stage white kinase is a polypeptide sequence consisting of five consecutive amino acid residues, and the sequence consisting of the amino acid with the highest frequency at each position in the sequence is LTPLK.

In summary, polypeptide phage display has been widely used in many biological research fields, and it is possible to screen phage peptides with clinical application value from polypeptide phage display libraries and then prepare a large number of polypeptides by synthetic, semi-synthetic, and other methods for clinical application. It can be predicted that the breadth and depth of polypeptide thallus display application in biological basic research and application development will be further expanded.