Library size

10⁷

Transformation required

Yes

Mechanism

Phage display systems can be grouped into two classes: true phage vectors and phagemid vectors. In both cases, the protein to be displayed is fused to the capsid protein.

Evolution

Typically the phage library screening entails several (usually three) consecutive rounds of panning and phage amplification before the selected phage and the polypeptide that they present are individually analyzed.

Protein displayed

Fv, scFv or Fab fragments

Proteins to be displayed

Soluble, non-toxic, compatible with crossing membranes

Surface anchorage

Capsid proteins

Properties of protein enhanced

Affinity, enzymatic activity, stability, folding, selection from cDNA libraries

Stability

Stable

Applications

• To enhance catalytic activity, proteolytic stability, or attach conjugation site and photoaffinity labels
• Affinity selection of protein fragments expressed from cDNA fragments
• Immunotherapy
• Developing diagnostic or therapeutic reagents in medicine
• Developing nanomaterials in material science
• Identifying receptor agonists or antagonists in cell biology

Advantages

• Biopanning of phage libraries on whole cells
• Wide range of pH could be used for selection

Disadvantages

• The low display rate can be a drawback. In the first round of panning where very few binders are to be enriched from a huge excess of unwanted phages, phages that are mostly “bald” can reduce the accessible molecular diversity of the library and the efficiency of the system
• If selection is done on cells, non specific phage binding is observed, due to phage protein interaction with cell membrane proteins
• In phage display, libraries must be transformed into bacteria, limiting the number of possible independent sequences to 10⁹–10¹⁰

Membrane protein research

Compatible

Nogo-66 - monotopic membrane protein (7,5 kDa)
ShuA - beta barrel forming protein, which pass through membrane 22 times (72.5 kDa)
MOMP - beta barrel forming protein, which pass through membrane 16 times (40 kDa)
Nef - peripheral protein (23 kDa)
Neuromodulin - peripheral protein (25 kDa)
Caveolin-1 - monotopic membrane protein (22 kDa)

References

1. Imai, S. et al. Development of an antibody proteomics system using a phage antibody library for efficient screening of biomarker proteins. Biomaterials 32, 162-169 (2011). 
2. Petrenko, V. Evolution of phage display: from bioactive peptides to bioselective nanomaterials. Expert Opinion on Drug Delivery 5, 825-836 (2008).

3. Paschke, M. Phage display systems and their applications. Applied Microbiology and Biotechnology 70, 2-11 (2005).
4. Reiersen, H. Covalent antibody display--an in vitro antibody-DNA library selection system. Nucleic Acids Research 33, 10 (2005).
5. Vithayathil, R., Hooy, R., Cocco, M. & Weiss, G. The Scope of Phage Display for Membrane Proteins. Journal of Molecular Biology 414, 499-510 (2011).
6. Morrison, K. & Weiss, G. Combinatorial alanine-scanning. Current Opinion in Chemical Biology 5, 302-307 (2001).
7. Clackson, T. & Wells, J. In vitro selection from protein and peptide libraries. Trends in Biotechnology 12, 173-184 (1994).
8. Galán, A. et al. Library-based display technologies: where do we stand?. Molecular BioSystems 12, 2342-2358 (2016).

Library size

10^13-14

Transformation required

No

Mechanism

DNA sequence of protein does not have a STOP codon and features a linker sequence at the C-terminus. Instead of detaching, the transcribed RNA and translated protein remain connected to the ribosome

Evolution

High affinity binders can be generated after 1^st round of selection, but most of the time, multiple rounds are done

Protein displayed

Antibody scFv fragments, "Designed Ankyrin Repeat Proteins"- DARPins, camelid nanobodies, DNA-binding proteins, receptors, membrane proteins

Proteins to be displayed

Most proteins including cytotoxic, chemically modified and membrane proteins

Surface anchorage

Ribosome

Properties of protein enhanced

Affinity, enzymatic activity (used rarely; water-in-oil emulsions have more advantages in this field), stability

Stability

Stable in Mg²⁺

Applications

Cancer treatment, antibody engineering, proteomics, diagnostics and therapeutics

Advantages

• Fast generation of big library as the complex is stabilized by Mg²⁺ and can be readily dissociated by the addition of EDTA
• A significant advantage of in vitro translation methods is the ability to modify the genetic code to allow the incorporation of non-canonical, unnatural amino acids, to give molecules with novel properties, such as cyclic peptides with increased serum, stability
• Rapid isolation and direct evolution of high-affinity functional proteins, particularly antibodies

Disadvantages

• By introducing mutations during PCR, a stop codon might appear in the middle of the sequence - the protein is unable to fold properly and mutant is lost. Moreover, RNA is less stable than DNA and requires an RNAse free environment, otherwise the majority of RNA will be degraded before reverse transcribed to DNA
•  In order to incorporate non canonical amino acids, DNA sequence has to be modified by introducing amber codons and specifically modified ribosomes have to be used
• Stem loops have to be added to the 5' and 3' ends, to stabilize RNA and a linker at the 3' end before the loops
• Polysomes can reduce the library size, because the protein is not formed and aggregates.

Membrane protein research

Incompatible. Membrane proteins are too hydrophobic and will not function if displayed. No pore forming activity or transfer activity would be evaluated

References

1. Lipovsek, D. & Plückthun, A. In-vitro protein evolution by ribosome display and mRNA display. Journal of Immunological Methods 290, 51-67 (2004).
2. He, M. & Khan, F. Ribosome display: next-generation display technologies for production of antibodies in vitro. Expert Review of Proteomics 2, 421-430 (2005).
3. Zahnd, C., Amstutz, P. & Plückthun, A. Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nature Methods 4, 269-279 (2007).

Library size

10¹⁰

Transformation required

No

Mechanism

Replication initiator protein (RepA) binds to the DNA template from which it has been expressed, a property called cis-activity. The protein of interest is binded to RepA protein C-terminus.

Protein displayed

Antibody scFv fragments

Proteins to be displayed

Soluble, including cytotoxic, chemically modified

Surface anchorage

In vitro

Properties of protein enhanced

Affinity, stability, resistance to degradation, longer half-life

Stability

Stable (>2 days)

Applications

For the selection of high affinity peptides and folded protein domains, including antibody fragments

Advantages

• Does not require any compartmentalization of the library-encoding nucleic acid
• The use of DNA to encode the displayed peptides provides advantages over RNA-based in vitro selection methods
• Very large libraries can be rapidly constructed and screened without separating transcription and translation steps and without purification of the protein–DNA complexes before selection
• Complexes do not require incubation under sterile or ribonuclease-free conditions
• Optimal ligands after 3-5 successive rounds

References

Odegrip, R. et al. CIS display: In vitro selection of peptides from libraries of protein-DNA complexes. Proceedings of the National Academy of Sciences 101, 2806-2810 (2004).

Library size

10¹⁴

Transformation required

No

Mechanism

Puromycin with a DNA linker is ligated to RNA after mRNA library generation. Translation is carried out and the puromycin molecule enters the P-site of ribosome. It takes the role of tRNA, which leads to the growing peptide chain being covalently connected to the puromycin molecule.

Evolution

To produce mRNA-displayed proteins requires ~3 days, to subject them to selection and evolution of enzymes for bond-forming reactions requires ~4-10 weeks

Protein displayed

Antibody scFv, RasIn1 and RasIn2 proteins

Proteins to be displayed

Soluble, including cytotoxic, chemically modified

Surface anchorage

In vitro

Properties of protein enhanced

Affinity, stability

Stability

Covalent

Applications

• Investigation of protein-protein interactions and the development and selection of peptides, enzymes, scFvs, and novel binders based on alternative scaffolds
• Protein evolution

Advantages

• Useful for performing in vitro selections in harsh environments that are not compatible with ribosome display. If the RNA stability is of concern in certain environments, the RNA can be replaced by its cDNA, as described in a variation of the mRNA display
• The mRNA display system allows libraries with sequence complexity approximately 10,000-fold that of phage display, 106-fold over yeast display or yeast two- and three- hybrid systems, and approximately 109-fold over colony screening approaches

Disadvantages

• Time consuming, technically demanding
• mRNA display has only been used for bond-forming enzymes (not to select enzymes that catalyze bond-breaking reactions or other covalent modification reactions)

Membrane protein research

Incompatible. Due to the limited expression of membrane-bound proteins by in vitro translation systems, mRNA-display cannot be utilized to address membrane protein related questions

References

1. Takahashi, T., Austin, R. & Roberts, R. mRNA display: ligand discovery, interaction analysis and beyond. Trends in Biochemical Sciences 28, 159-165 (2003). 
2. Seelig, B. mRNA display for the selection and evolution of enzymes from in vitro-translated protein libraries. Nature Protocols 6, 540-552 (2011). 

3. Cetin, M. et al. RasIns: Genetically Encoded Intrabodies of Activated Ras Proteins. Journal of Molecular Biology 429, 562-573 (2017).
4. Lin, H. & Cornish, V. Screening and Selection Methods for Large-Scale Analysis of Protein Function. ChemInform 34, (2003).

Library size

10⁷

Transformation required

No

Mechanism

A fusion protein - P2A and an scFv antibody bind to the same molecule of DNA from which it has been expressed. Following in vitro coupled transcription and translation, the P2A protein makes a covalent link between scFv genotype and scFv phenotype, by producing a stable protein–DNA complex

Evolution

Fast cycle time: in a few hours, unique scFvs can be enriched, isolated and directly amplified for the next rounds of selection

Protein displayed

Antibody scFv fragments

Proteins to be displayed

Developed for antibody display

Surface anchorage

In vitro

Properties of protein enhanced

Affinity selections

Stability

Covalent

Applications

Advantages

• scFv can be genetically fused to the P2A protein creating the smallest imaginable antibody selection particle: a protein and its gene
• CAD is the only antibody selection method providing a direct covalent link between an scFv gene and its protein, increasing the chemical stability of the panning complex

Disadvantages

Product inhibition of uncomplexed fusion proteins without DNA

Membrane protein research

Incompatible - only used for antibody display and no membrane proteins can be evolved with this system

References

Reiersen, H. Covalent antibody display--an in vitro antibody-DNA library selection system. Nucleic Acids Research 33, 10 (2005).

Library size

Up to 10¹⁴

Transformation required

Yes

Mechanism

The most common yeast display system employs fusion of the protein of interest to the C-terminus of the Aga2p subunit. Induction of protein expression results in surface display of the fusion protein through disulfide bond formation of Aga2p to the β1,6-glucan-anchored Aga1p domain of agglutinin. The epitope tags allow quantification of fusion protein expression, and thus normalization of protein function to expression level by flow cytometry using fluorescently labeled antibodies

Protein displayed

Antibody scFv fragments, Fab, single-chain T cell receptors, major histocompatibility complex (MHC)

Proteins to be displayed

Soluble and membrane, nontoxic, compatible with crossing membranes

Surface anchorage

Agglutination proteins, flocculation proteins

Properties of protein enhanced

Affinity, specificity

Applications

• Indispensable and efficient way for affinity maturation in antibody engineering
• A method to immobilize enzymes and pathogen-derived proteins for vaccine development  

Advantages

• Yeast as eukaryotic microorganism owns post-translational modifications as well as processing machinery and conditions homologous to the mammals
• Possesses eukaryotic machinery to express, assemble and export onto the surface both monomeric and oligomeric proteins in a fully native-like state
• In a direct comparison with phage display using the same antibody library and target, the yeast system sampled the library repertoire more comprehensively
• Compatibility with flow cytometric analysis, which allows quantitative measurements of equilibrium binding constants, dissociation kinetics, stability, and specificity of the displayed proteins without the laborious requirements of soluble protein expression and purification

Disadvantages

The potential drawbacks are similar to phage display in that the expression of extracellular proteins is favoured and affinity maturation is complicated by avidity effects due to the multiplicity of the displayed peptides or proteins on the cell surface

Membrane protein research

Compatible

References

1. van Rosmalen, M. et al. Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning. Journal of Biological Chemistry 292, 1477-1489 (2016). 
2. Gai, S. & Wittrup, K. Yeast surface display for protein engineering and characterization. Current Opinion in Structural Biology 17, 467-473 (2007). 
3. Bowley, D., Labrijn, A., Zwick, M. & Burton, D. Antigen selection from an HIV-1 immune antibody library displayed on yeast yields many novel antibodies compared to selection from the same library displayed on phage. Protein Engineering, Design and Selection 20, 81-90 (2007).     
4. Shusta, E., Pepper, L., Cho, Y. & Boder, E. A Decade of Yeast Surface Display Technology: Where Are We Now?. Combinatorial Chemistry & High Throughput Screening 11, 127-134 (2008).

Library size

10⁷

Transformation required

Yes

Mechanism

For the purpose of displaying foreign proteins on the surface of baculovirus particles as well as on infected insect cells, gp64 serve as a fusion partner that together with a chosen target protein gets incorporated into the cell membrane

Proteins to be displayed

Soluble and membrane, nontoxic, compatible with crossing membranes

Surface anchorage

Agglutination proteins, flocculation proteins

Properties of protein enhanced

Increased binding affinity and improved catalytic properties

Applications

• Therapeutics
• Eukaryotic surface display libraries based on the baculovirus system allow selecting for specific binding proteins while providing post translational modifications
• Baculoviruses are non-pathogenic to mammals and thus, are an ideal carrier for antigenic epitopes or proteins intended to induce neutralizing immune response when administered as vaccines
• Eukaryotic expression libraries are a powerful tool for a variety of applications such as finding new ligands, identification of cellular interaction partners and affinity maturation of antibody and antibody fragments

Advantages

• The mammalian expression system is inherently competent for processing and secreting eukaryotic proteins. Consequently, mammalian cell display offers vast potential to display full-length human antibodies;
• Insect cells might be more feasible to express complex protein structures than e.g. yeast;
• As compared to mammalian cell lines, insect cells such as Sf9 and T. ni cells may be considered as a more robust and efficient display scaffold, since handling is less laborious, less costly and target proteins are incorporated more efficiently and homogeneously into the cellular membrane;
• Selection of specific clones by FACS is a fast and attractive method and does not require the cells to stay intact, but instead baculoviruses can be recovered by filtration and be used for re-infection

Disadvantages

An obvious challenge remains to be solved that relatively low transformation efficiency of mammalian cells diminishes the actual repertoire size in contrast with phage display, leading to unlikely straightforward isolation of  antibodies with remarkable affinity. Moreover, the mammalian cell proliferation rate is slower, and such cells require more specific culture conditions in vitro than microbial cells. These drawbacks necessitate great efforts to improve the mammalian cell display platform.

Membrane protein research

Compatible

References

Oker-Blom, C. Baculovirus display strategies: Emerging tools for eukaryotic libraries and gene delivery. Briefings in Functional Genomics and Proteomics 2, 244-253 (2003).

Library size

10¹⁰

Transformation required

No

Mechanism

Experimental conditions are adjusted so that, in most cases, one compartment contains one DNA molecule. The DNA fragments encode fusion proteins containing a DNA-methyltransferase, which can form a covalent bond with a 5-fluoro deoxycytidine base at the extremity of the DNA fragment. The resulting library of DNA–protein fusions is extracted from the emulsion and DNA molecules displaying a protein with desired binding properties are selected from the pool of DNA–protein fusions by affinity panning on target antigens.

Typical enrichment factor per round

>1000

Protein displayed

In vitro compartmentalization  using water-in-oil emulsions has been used for the selection of peptide ligands and for the directed evolution of DNA methyltransferases, bacterial phosphotriesterase, Taq polymerase, luciferase and human telomerase.

Proteins to be displayed

-

Surface anchorage

-

Properties of protein enhanced

Enzymatic activity, thermal stability

Stability

Stable

Applications

Protein evolution, to enhance their enzymatic activity, and thermal stability.

Advantages

Since DNA is added at limiting dilution, each aqueous droplet contains a single gene, and acts as a unique, independent reaction vessel

Disadvantages

• Many proteins of interest, in particular more complex, multidomain proteins are more efficiently expressed in eukaryotic expression systems
• More than one DNA molecule might be present in one water compartment of the water-in-oil emulsion, which would lead to the incorrect assignment of genotype and phenotype and, therefore, to the selection of DNA molecules coding for non-binding proteins

Membrane protein research

Incompatible. Membrane proteins cannot be reconstituted into surfactant layer of w/o emulsion

References

1. Ghadessy, F. & Holliger, P. A novel emulsion mixture for in vitro compartmentalization of transcription and translation in the rabbit reticulocyte system. Protein Engineering, Design and Selection 17, 201-204 (2004).
2. Bertschinger, J. & Neri, D. Covalent DNA display as a novel tool for directed evolution of proteins in vitro. Protein Engineering Design and Selection 17, 699-707 (2004).

Library size

10⁷

Transformation required

No

Mechanism

Membrane protein is inserted to lipid bilayer. Protein can insert by himself or with the help of BAM complex and additional helper proteins

Evolution

Single round of selection can be achieved within 1 day

Protein displayed

Alpha-hemolysin pore forming protein, G protein-coupled receptor, caveolin

Proteins to be displayed

Soluble and membrane, non-toxic, toxic, compatible with crossing membranes

Surface anchorage

Membrane proteins

Properties of protein enhanced

Pore-forming activity, enzymatic activity, ...

Stability

Stable

Applications

Membrane protein evolution, therapeutics

Advantages

• In vitro translation system produces proteins without using living cells. One DNA sequence can be trapped in one liposome and toxic, bacterial growth inhibiting protein can be translated
• Rapid and efficient mutant selection
• Proteins that affect cell growth can also be engineered because the protein is synthesized in vitro
• Conditions appropriate for the target membrane protein can be adopted because the lipid composition of the liposomes is adjustable and internal solution can be adjusted as needed
• No membrane proteins other than the target one are present

Disadvantages

Size variety of liposomes reduce enrichment factor if multiple genes are trapped inside one big lisopome

Membrane protein research

Alpha-hemolysin was translated inside liposome, self-inserted to membrane and formed pore. By doing evolution of this protein, mutants showing better pore forming and self-inserting properties were selected by FACS

References

1.Fujii, S., Matsuura, T., Sunami, T., Kazuta, Y. & Yomo, T. In vitro evolution of  alpha-hemolysin using a liposome display. Proceedings of the National Academy of Sciences 110, 16796-16801 (2013).
2.Goodsell, D. & Olson, A. Structural Symmetry and Protein Function. Annual Review of Biophysics and Biomolecular Structure 29, 105-153 (2000).