Unnatural Amino Acids for Phage Display Insertion

Phage display technology fuses exogenous peptides and phage capsid proteins and displays them on the phage surface, performs high-throughput screening and enrichment, and conducts qualitative analysis on clones with required functions. The technology displays antibodies, antibody fragments, peptides, cDNA, etc. Phage peptide library construction is an important biotechnology tool for screening peptide sequences with specific functions. This process usually starts from the phage display theory, using phage (such as M13 phage) as a carrier to display random peptide sequences on the phage coat protein. Phage peptide library technology is widely used in many fields such as antibody engineering, drug discovery, protein-protein interaction research, etc. It provides a powerful and efficient method to identify and optimize peptide sequences with specific biological activities.

Phage Definition

Phages are a type of virus that can infect bacteria. They can be divided into multiple genera according to their host range and infection characteristics. Common phages include single-stranded DNA phages (such as M13, which is also a filamentous phage), double-stranded DNA phages (such as T4, T7, λ), etc. Among them, M13 phage is often selected for the display of exogenous peptides or proteins, mainly because it has the following characteristics:

• Single-stranded DNA structure: The genome of M13 is single-stranded DNA, which is easy to operate and sequence in the laboratory. This simple structure allows scientists to easily insert exogenous DNA and construct a phage display library.
• Easy to find infected cells: M13 phage can bind to host bacteria through the cilium structure (Pilus) on the cell surface and infect cells. This binding method is relatively specific, allowing scientists to selectively infect specific bacterial strains.
• High stability: M13 phage particles are very stable and can maintain integrity under conventional experimental conditions. This stability ensures the reliability and repeatability of phage display technology.
• Easy to amplify: After infecting bacteria, M13 phage can quickly replicate itself and release it into the culture medium, which is convenient for large-scale production.
• Friendly phage display: The outer coat proteins (pIII, pVIII, etc.) of M13 phage can be used to display exogenous peptides or proteins, which is convenient for phage display technology to screen antibodies, drugs or protein interactions.

Therefore, the display technology based on M13 phage is widely used in protein engineering, antibody screening, vaccine design and other fields, providing a powerful tool for biomedical research.

Phage Display

Phage display technology is a molecular biology technology. Through genetic recombination, the gene fragment encoding the foreign peptide or protein is precisely inserted into the gene of the phage coat protein, so that the foreign peptide or protein can be fused with the phage coat protein and maintain its specific spatial conformation and biological activity, so as to be displayed on the phage surface. These displayed foreign peptides or proteins can have specific affinity with the immobilized target protein. In this way, peptides or proteins with specific binding properties can be enriched and screened. Ultimately, phage display technology can help researchers obtain peptides or proteins with specific functions for various biological research and applications. Phage display technology has the following advantages:

• The binding activity of phages with other molecules is unified with the amplification of phages, making the phage display library an efficient screening system.
• Through the unique mutation function of biological systems, a mutated display structure is generated, and the mutated structure may contain fragments with improved affinity to the target molecule.
• It has been successful in obtaining diversified human monoclonal antibodies with high affinity, and has become the third generation of antibodies after polyclonal antibodies obtained from immunized animals and monoclonal antibodies obtained by hybridoma technology.
• In screening ligands, phage libraries have the advantages of rich resources and rapid screening.
• When the structure of the target molecule is unclear, the target molecule is difficult to purify, or its conformation may change once purified, the ligand can still be found through screening strategies.

Phage Display Library

Phage Display Library is a biotechnology tool used to study protein-protein, protein-peptide and protein-nucleic acid interactions. It is achieved by displaying variants of peptides or proteins on the surface of bacteriophage (a virus that infects bacteria). This technology was first developed by George P. Smith in the late 1980s and has been widely used in subsequent research. The core concept of phage display library is to insert target DNA fragments into the phage genome so that the amino acid sequences encoded by these DNA fragments can be displayed on the outer surface of the capsid protein of the phage. In simple terms, researchers first construct a DNA library containing a large number of different DNA sequences, and then transfer these sequences into the phage genome through phage display technology to generate a library containing a variety of different phage particles. Each phage particle displays a specific peptide or protein variant on its surface.

Phage Display Library Construction

At present, the main methods for inserting non-classical amino acids into phage-displayed peptide sequences can be roughly divided into chemical methods and biological methods. The specific strategy should also be explored from the process of displaying peptides on phage capsid proteins. The construction of phage random peptide library is to integrate exogenous complementary DNA (cDNA) into viral DNA using recombinant DNA technology to synthesize phage plasmid library. Then transform it into Escherichia coli to synthesize bacterial library, and infect auxiliary phage at the same time, so that the peptide is displayed on the capsid protein of phage, and the synthesized phage library is used for biological selection. In this process, to think about inserting non-natural amino acid sequences into peptide chains, we must first consider how natural amino acid sequences are transcribed, translated, and expressed on phage capsid proteins. There are a total of 64 codons for translating and expressing 20 natural amino acids, in addition to 3 stop codons UAA, UAG, and UGA.

Fig. 1. Bacteriophages are involved in replication and translation.

As shown in Fig. 1, after phage invades Escherichia coli, it mainly participates in two processes: replication and translation. Single-stranded DNA must first be reverse transcribed into double-stranded DNA. Then it is continuously replicated and becomes single-stranded DNA. All these processes require the participation of enzymes, which are derived from: single-stranded DNA is transcribed into mRNA, and mRNA is translated into some key proteins. At the same time, mRNA will also translate into capsid protein, and then the assembly of capsid protein and single-stranded DNA will form the M13 phage, which will then leave the bacteria. In the translation process of capsid protein and display peptide, the codon on mRNA and the anticodon on tRNA responsible for carrying amino acids are recognized through base complementary pairing, decoding the genetic information on nucleic acid into amino acid information on the protein peptide chain. The process of tRNA loading amino acids requires catalysis by aminoacyl tRNA synthetase (aaRS), which covalently binds the amino acid to the 3' end CCA tail of tRNA.

Phage Display with Unnatural Amino Acids

According to the translation process and display results of proteins/peptides, some biological and chemical methods have been developed through design to achieve the insertion of non-natural amino acids into the peptide sequences displayed by phages, or to achieve the diversity of displayed peptides by modifying the residues of amino acids after translation. The main methods are:

(1) Designing tRNA corresponding to the stop codon and aminoacyl-tRNA synthetase (aaRS) carrying non-natural amino acids;

(2) Designing close structural homologs to replace the 20 common amino acids in nutritionally deficient strains;

(3) Modification of residues after translation;

(4) Designing tRNA that recognizes four codons.

* Features of unnatural amino acid products:

• tRNA/aaRS Pair

The translation of proteins/peptides includes three stages: initiation, elongation, and termination. The site-specific insertion of non-natural amino acids into the peptide chain is mainly carried out in the elongation stage. In this stage, aminoacyl tRNA synthetase (aaRS) recognizes the amino acid it catalyzes and connects the amino acid to the ribose at the 3' end of tRNA by forming an ester bond to form aminoacyl tRNA. Each of the 20 natural amino acids has its corresponding specific aminoacyl tRNA synthetase. tRNA contains an anticodon that can recognize the codon in mRNA, so the next step is that aminoacyl tRNA can bind to the corresponding site of mRNA and connect the amino acid it carries to the carboxyl end of the existing peptide chain. Using the orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pair, the insertion of non-natural amino acids into random peptides of phages displayed in the Escherichia coli host requires several conditions:

(1) A new tRNA must be constructed: the new tRNA cannot be recognized by the existing Escherichia coli aminoacyl-tRNA synthetase, and it must also be able to function effectively in translation. The tRNA must not bind to codons encoding any of the 20 classical natural amino acids, and deliver unnatural amino acids by binding to nonsense codons or four-base codons. Nonsense codons can be used with inhibitory tRNAs for mutagenesis of conventional protein polypeptide sequences; four-base codons can be effectively bound by tRNAs containing modified anticodon loops.

(2) A new aminoacyl-tRNA synthetase is required: the new synthetase can aminoacylate its orthogonal tRNA, but cannot recognize any endogenous E. coli tRNA. This synthetase must aminoacylate tRNA only with the desired unnatural amino acid, without using the classical 20 amino acids. Likewise, unnatural amino acids cannot serve as substrates for endogenous synthetases.

(3) Unnatural amino acids must be able to be efficiently transported into the cytoplasm.

• Chemical Modification

As shown in Fig. 2, unique reaction handles can be displayed on phage-displayed peptides, such as seleno groups (generally unstable), azide, cysteine thiol groups, and phenolic hydroxyl groups. Among them, azide is inserted by removing all methionines and utilizing the electron isostericity of azide homologous alanine and methionine. Among the residues in the natural amino acid sequences of proteins and peptides, some have certain reactivity, and some reactive functional groups can also be introduced by non-natural amino acids. For example, cysteine residues can participate in Michael addition, nucleophilic substitution, and oxidative dehydrogenation to synthesize olefins; alanine participates in the ring-closing reaction of isocyanide; lysine participates in the amidation of active esters; arginine participates in the ring-closing reaction of aldehydes and ketones, etc. In some reports, reactive phage-displayed peptides have unique aldehyde functional groups that can participate in some reactions to perform post-translational modifications on random peptides. The aldehydes here are obtained by oxidation of N-terminal serine/or threonine residues with NaIO4.

Fig. 2. Phage chemical modification (ACS Chem Biol. 2012, 7(1):123-38).

Phage display technology is a powerful molecular biotechnology for high-throughput target-ligand screening, which has good applications in many areas, such as target discovery, drug discovery, epitope discovery (new routes for diagnosis and vaccines), screening of DNA binding proteins, etc. Phage display technology can be used to perform affinity screening on targets based on fixed targets, cell-based targets, in vivo screening of animals, in vitro target screening of tumor resection, and in vivo target screening of human sources. It is flexible and diverse in the research of disease-related protein targets and ligands, and can better screen out active ligands. At present, a variety of antibody drugs determined by phage display technology have been successfully marketed for disease treatment, such as Adalimumab, Belimumab, Rexiba-cumab, Ranibizumab, Ramucirumab for antibodies; Romiplostim, Ecallantide, Regine-satide for peptides, etc.

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