Team:TU Darmstadt/Project/Background

TU Darmstadt


Background


Virus-like Particles

Virus-like particles (VLPs) are nanoparticles formed by self-assembly of viral envelope or capsid proteins.[1]Although they are structurally similar to authentic viruses, VLPs lack any DNA or RNA. They are capsids made of protein subunits and unlike viruses, do neither have nucleic acids nor a viral life cycle, allowing their safe usage for many therapeutic applications. They are still capable of entering target cells.[2] VLPs can be used to stimulate a strong immune response by displaying a high density of epitopes. VLP production is straightforward and has been demonstrated for a variety of expression systems.[3] In contrast to other nanoparticles like liposomes VLPs are way more stable and are not as restricted regarding packaging capacity.[4]

Structure

VLPs are structurally and functionally diverse. They are roughly classified in non-enveloped or enveloped. Enveloped VLPs are surrounded by lipid layers embedded with glycoproteins derived from membranes of host cells. Each category is further subdivided in single- or multiple-capsid protein VLPs. Most VLPs have an icosahedral geometry, others structures are spherical or filamentous. Their size range is similar to those of viruses with 22-150 nm. [5]

Expression

As stated above VLPs can be produced in a variety of expression systems. The choice of the best system depends on the structure of the VLP and whether a large-scale production is pursued or not. For example, they can be produced in a baculovirus system, in mammalian cells, bacterial cells or yeast cells. The production in insect cells has the advantage of correctly folded antigens, resulting in improved immunogenicity if used for vaccines, and high-density cell cultures, which can easily be scaled up. As the baculovirus system is quite complicated to regulate and relatively slow, other expression systems are often used.[5] Structurally simple non-enveloped VLPs can easily be produced in yeast or Escherichia coli. Another method is the mammalian vaccinia-virus-based expression system. Even though this system is used for VLP preparation it is not very often used for production of VLPs. Yeast, insect or mammalian systems can post-translational modify VLPs, which also enhances immunogenicity. [6] Last but not least, it is also possible to produce VLPs in a cell free assembly system. In the end, the choice of expression system affects the downstream purification procedure. [3]

Purification

Recombinant, in vivo synthesized VLPs have to be purified. Generally the first step of purification is the transfer of the VLP to the surrounding medium or solution. This can be done by cell lysis. The lysis step is not required if the VLP is expressed in a cell system that ensures secretion like mammalian and insect cells. For protection and further purification the extraction buffers are often complemented with chelating and reducing agents. Furthermore protease inhibitors and nucleases are often added. In the second step, the cells and cell debris have to be removed. This can be done by a combination of ultracentrifugation and size-exclusion chromatography. After this the VLP-solution has to be further purified and concentrated. Some VLPs are stable enough to be precipitated with PEG or/and ammonium sulfate. In laboratory scale ultracentrifugation in CsCl or sucrose gradients is often sufficient as purification for most applications. In industrial scale chromatographic processes are prefered as ultracentrifugation lacks scalability. [3]

Application

In non targeted drug delivery therapies, the agent is often evenly distributed throughout the entire body. VLPs can be used to function as a platform for targeted drug delivery through the attachment of cell specific ligands or antibodys, reducing the required dose and following burden on the recipient. [4] Subunit vaccines can suffer from poor presentation to the immune system, incorrect folding of the target protein and from having a poor immunogenicity. In this regard, VLPs have shown a very high effectiveness as candidate vaccines. To date VLPs have been produced for vaccines against more than 30 different viruses that infect humans and animals. [7] Commercialised VLPs help e.g. to protect humans from hepatitis B virus. Additionally VLPs can be used as nanocontainers in gene therapy and as new nanomaterial in the area of bio nanotechnology. [3] For example the growth behavior of semiconducting nanocrystals like CdS can be controlled inside VLPs. [8] Other iGEM Teams like Wageningen (2012) and Lethbridge (2018) also realized the potential of VLPs. While Wageningen used capsids from other viruses, for example hepatitis B and Cowpea Chlorotic Mottle Virus (CCMV) to create a modular platform with noncovalent interactions [10] , Lethbridge specialized on P22 capsids. They wanted to establish a toolkit for delivery cargos. [11]

P22 VLP

Origin

The P22 VLP originates from the temperate bacteriophage P22. Its natural host is Salmonella  typhimurium. Since it was isolated half a century ago it has been characterized thoroughly and has become a paradigm system for temperate phages. To date, nearly everything is known about its lifecycle including virions. Because of that and its specific properties it generates an accessible VLP platform. [9]

Structure

An assembled P22 VLP consists of 420 copies of coat protein (CP) and 100 to 300 copies of scaffold protein (SP). [12]
The shell of the VLP is formed by the 46.6 kDa CP. The coat protein occurs in one configuration, which contains a globular structure on the outer surface and an extended domain on the inner surface. Seven CPs arrange in asymmetric units, which form the icosahedral structure of the VLP. [13]
The 18 kDa SP is required for an efficient assembly and naturally consists of 303 amino acids. It has been shown, that an N-terminal truncated SP of 163 amino acids retains its assembly efficiency. The 3D-structure is composed of segmented helical domains, with little or no globular core. In solution is a mixture of monomers and dimers present. [14]
When purified CPs and SPs are mixed, they self-assemble into VLPs.

Figure 1: Assembly of the VLP

Characteristics

P22 VLPs occur as a procapsid after assembly. If the VLP is heated up to 60 °C, the CP rearranges, forming the expanded shell form (EX). This form has a diameter of about 58 nm and the volume is doubled compared to the one of the procapsid. The expanded shell form changes into the whiffleball form (WB) when heated further up to 70 °C. The whiffleball has 10 nm pores, while the procapsid or the expanded shell form only have 2 nm pores. [15] Furthermore, the P22 VLP consists of SP and CP, but it can also assemble with CPs only. If it assembles without SP it can form two sizes of capsids. The small capsid is built as a T = 4 icosahedral lattice with a diameter between 195 Å and 240 Å. The larger capsid also has an icosahedral lattice, but it is formed as T = 7. T being the "triangulation number", a measure for capsid size and complexity. [38] Moreover, it is like the wild type VLP, which includes the SP. The diameter of the wild type VLP, is between 260 Å and 306 Å. Each capsid consists of a 85 Å thick icosahedral shell made of CP. [16]

P22 VLP as a model

We decided to work with P22 because it has several big advantages:

First of all, it is easy to express. The bacteriophage P22 infects Enterobacter, for example E.  coli. [4] Therefore, the codon usage of the structural genes is already optimized for E. coli. Farther E. coli is a fast growing, inexpensive and well known host for heterologous gene expression. On top of that there is no need in synthetic modification of CP and SP after expression.[17] The VLP assembles spontaneously.

Secondly P22 VLPs assemble in vivo and in vitro in a correct form.[18] The in vitro assembly helped a lot in research. The assembly and modification of our VLPs got tested in defined conditions. This way we could learn a lot about the mechanism and we proved our concept. The disadvantage of in vitro assembly is the high effort. All Proteins need to be expressed and purified separately. With our modular platform we wanted to keep the usage of VLPs as simple as possible. With in vivo assembly the proteins all get expressed at once and assemble within the cell. This way we only need to purify the finished VLP once at the end of the process.

A really big advantage is the easy modification of the P22 VLP. The C-terminus of each CP points towards the outside of the VLP. So, by expressing an additional tag at this end, proteins can attach to this site and are presented on the outer surface. The conformation of CP is not impaired by this modification. Hence, the conformation of the whole VLP is also undisturbed. [17] It is possible to modify more than one CP in a VLP. This way it is possible to achieve a diverse surface functionalization by fusing different proteins to the C-termini.

Since we are talking about big advantages. A literally big advantage is the size of the P22 VLP. The huge diameter allows to store big proteins or even protein complexes inside. [19] The storing of cargo inside the VLP can be achieved in different ways. One way is a simple encapsulation of the cargo. Here the VLP assembles around the molecule of interest. Another possibility is the fusion of proteins to the SP. After the assembly the scaffold protein is on the inside of the VLP. This way the SP works as a specific encapsulation signal. [18]
The cargo for VLPs is not restricted to proteins. It can also be DNA, RNA, or everything fitting inside.
In the end, there are more than these opportunities to modify a P22 VLP. As an example, one more way is the modification with viral decoration proteins, which bind at symmetry specific sites on the capsid. [20]

Sortase

Transpeptidase: Sortase

Sortases belong to the class of transpeptidases and are mostly found in gram-positive bacteria. The high rate of resistance to several antibiotics targeting gram-positive bacteria is also based on the property of this enzyme class. Sortases can non-specifically attach virulence and adhesion‐associated proteins to the peptidoglycans of the cell-surface. [21, 22]
In general, sortases are divided into six groups (A-F) that have slightly different properties and perform three tasks in cells. Group A and B attach proteins to the cell-surface while Group C and D help building pilin-like structures. Group E and F are not properly investigated yet which is why their exact function is not known. [23] For our project we are especially interested in the sortases of the group A since they covalently attach various proteins or peptides on the cell membrane as long as their targeting motif is at the C-terminus of the corresponding protein. In comparison to other transpeptidases Sortase A has the advantage that it is rather stable regarding variations in pH [24] and temperature. [22] Sortase A catalyzes the formation and cleavage of a peptide bond between the C-terminal LPXTG amino acid motif and an N-terminal poly-glycine motif. The enzyme originates from Staphylococcus aureus and is able to connect any two proteins as long as they possess those matching target sequences. [25] In the pentapeptide motif LPXTG, X can be any amino acid except cysteine. [21] Sortase A is rather promiscuous with regard to the amino acid sequence directly upstream of this motif, a fact that makes it optimal for labeling applications. Even better, amino acids C-terminal of the poly-glycine motif are not constrained to a certain sequence. [26] The reaction works with any primary amine. [30]

Reaction

To better understand how the enzymatic reaction works it is necessary to look at the crystal structure of Sortase A. The enyzme consists of an eight-stranded β‐barrel fold structure. The active site is hydrophobic and contains the catalytic cysteine residue Cys184 as well as a key histidine residue H120 that can form a thiolate-imidazolium with the neighboring cysteine. An additional structural property that also other sortases show is the calcium binding site formed by the β3/β4 loop. The binding of a calcium ion slows the motion of the active site by coordinating to a residue in the β6/β7 loop. This helps binding the substrate and increasing the enzymatic activity nearly eightfold. [26] When a substrate gets into the active site, the cysteine attacks the amide bond between the threonine and the glycine in the LPXTG motif. After this the protonated imidazolium serves as an acid for the departing glycine with unbound NH2 of the former amide bond while the rest of the motif is bound to the cysteine residue. Another glycine nucleophile is then necessary in its deprotonated form to attack the thioester and re-establish an amide bond at the LPET-motif. This reaction is dead-ended if the used nucleophile is water.[26] Due to the fact that the mechanism is based on protonated forms of the catalytic residues the reaction is quite pH-dependent. Although the Sortase A in general is relatively stable between pH 3 and 11 the reaction works best around pH 8.[28]

Figure 2: Sortase reaction mechanism

Sortase variants

Due to the fact that the wildtype Sortase A shows rather slow kinetics, a pentamutant has been developed (Sortase A5M). [29] This version of the enzyme carries mutations in P94R/D160N/D165A/K190E/K196T which lead to a 140- fold increase in activity. Thereby, reaction rates are improved even at low temperature, however, Sortase A5M is still Ca2+-dependent. This dependence interferes with potential in vivo usage, as the concentrations of calcium in living cells can vary considerably. Hence a sortase mutant that acts across high differences in calcium concentrations or even works completely Ca2+-independently would be required for in vivo applications of sortase. To attain a high yield enzyme which is also calcium-independent Ca2+-independent mutations were combined with the Sortase A5M resulting in Sortase A7 variants such as the Sortase A7M. The newly achieved calcium-independence of these variants enable sortase applications not only in vitro but in vivo as well. [30]

Sortase A7M

For our project we chose to work with this optimized Sortase A7M. Its size is about 17.85 kDa and it has been shown to be stable for several weeks in the fridge at 4 °C. It also possesses the same properties of pH stability like other sortases [31] but comes with the advantage of being calcium independent. [32] "Sortagging" applications have included the cyclization of proteins and peptides [31] , modification and labeling of antibodies and the synthesis of protein conjugates with drugs, peptides, peptide nucleic acids and sugars. [27] Moreover it poses a lot of advantages for the binding of two proteins in vivo since it has relatively small tags which avoids putting too much metabolic burden on the cells when expressing the proteins of interest. This also avoids disturbing the folding of the proteins of interest and the later biological functions since the Sortase A7M is able to work under physiological conditions. [30] Other methods like the intein- based labeling of surfaces require large fusion-proteins with the intein domain which puts stress on the living cells and might cause folding and solubility issues. Another application for sortase-mediated systems is the anchoring of proteins on the cell wall of gram-positive bacteria which can be used for display of heterologous proteins. [26] It is also possible to attach non-biological molecules to the respective tag. The accessibility and flexibility determine the ability of a sortase enzyme to recognize the sorting motif and catalyzing the transacylation.

Why Modularity?

Nowadays, flexibility is one of the key qualities in nearly all domains of science.
A modular platform is well-suited to be created with Virus-like particles (VLPs), as VLPs have many characteristics which allow different applications. Firstly, VLPs can be modified on the outer surface or inner cavities enabling different opportunities for applications. Secondly, viruses and bacteriophages play a huge role in biological systems [33] and can be produced in heterologous hosts. Each type of virus or VLP is uniform and biocompatible. Furthermore, VLPs are easily functionalized. [3]

Potential applications

A significant characteristic for modularity is the amount of different possible applications. VLPs can be used as vaccines For this, the VLP is surface-modified with the relevant antigen. [34] Another opportunity of application is drug delivery, more specifically targeted drug delivery. It can be used for localized diseases, like cancer. [33] It is also possible to encapsulate enzymes. If the enzymes are in the interior of the VLPs, they are protected against enzymatic degradation and denaturation in organic solvents. The encapsulated enzymes can be seen as nanoreactors, which are used in catalytically active soft nanomaterials. Moreover, the activity may be enhanced and multienzyme structures can be immobilized. As a result, the VLP system is very modular. [15]

Advantages of modularity


All of these applications are important and pioneering. But until now, it is quite expensive to develop any one of them, since it takes a lot of time and research to clone all needed parts in one or more genetic constructs.
To remedy this, we created the modular platform for Virus-like particles, the real MVP. The only thing left to be cloned by any user of our system is their protein of interest. So instead of the time consuming cloning of multiple fusion-proteins the approach is simplified to just adding a prefix to the protein of interest through overhang-primers. This streamlining of the process could save lifes by getting medicine faster to people in need.

Figure 1: Cloning with MVP saves time, by simplyfying the cloning from creating fusion proteins to simpler pre-/suffix addition.

It is also less expensive, needing less work hours and a reduced need in lab equipment. By reducing the research costs, the product gets cheaper. That is obviously great for any kind of application. But it is particularly important when it comes to medical treatment.

Figure 2: The MVP saves money by reducing lab equip and research cost.


During the interview with Dr. Schülke, we learned that a lot of testing is necessary, if the VLP is to be used for any kind of medical application. By using our modular platform, the testing for a new application scenario can be shortened, as only the modified VLP would have to be analyzed. Therefore, time and money could be saved.

Figure 3: Since only new parts need to be tested, using existing parts can save a lot of time and labor.

Our modular platform is easy to use, since all needed proteins are already cloned into the backbone. In future the functionalization of the surface can be easily adjusted by varying the ratio of modifiable and unmodifiable coat, which means coat protein with and without LPETGG-tag. The ratio can be adjusted with no effort then, because different promoters will be used in the vector. This is the principle of dual expression.
With our modular platform the Virus-like particle is functionalized after the assembly of coat and scaffold protein. This way the protein of interest cannot disturb the assembly process. The consequence is a higher yield of fully assembled functionalized VLP.
With the real MVP, everybody gets access to the advantages of Virus-like particles.
Want to know more about applications? Click here.

Conclusion

Within the recent decades, Virus-like particles experienced an increase in popularity due to their outstanding performance in various fields. This popularity can mainly be attributed to the fact that they are highly customizable. The regular structure of capsids allows for precise modification that is unmatched by other nanomaterials (e.g. inorganic or lipid based ones).[3] For example, vaccinations with modified capsomers have shown to increase the immune response and enhance the efficiency of the vaccine as shown.[35] Cell specific drug delivery using capsomers has also shown to be really promising and has already been proven to be effective.[36] Most displayed molecules usually require covalent attachment to the surface of the VLP. So far, this has either been realized by genetic fusion to the protein coding sequence of the monomers, through "click" chemistry or by conjugation to surface presented cysteines and lysines.[4] An easy to use, modifiable platform based on covalent linkage could prove as a valid alternative. Such a platform could save research groups huge amounts of time and render many more promising projects affordable.

The P22 bacteriophage capsid is one of the well documented and characterized capsomers. Recent research by Patterson et al.[17] on the P22 capsid that inspired us to use it in our iGEM project. Especially the fact that its monomers show considerable tolerance towards being fused with various proteins of interest[37] encouraged us to work with it. Some studies have shown[36] however that there are restrictions to the repertoire of proteins one can genetically fuse to the monomers, since it can hinder the VLP assembly.

The system we are using is based on the covalent linkage of arbitrary, tagged proteins to a complementary tag at the surface exposed C-terminus of the P22 coat protein monomer using an enzymatic reaction, thus it should be able to work around some of these restrictions. People using our tagged VLPs only need to add the respective tag to their protein of interest in order to link proteins to the P22 capsomer. Excessive and time-consuming cloning of multiple fusion proteins is no longer necessary and gets replaced by simply adding a short tag as a pre- or suffix sequence to the proteins of interest. Once purified, these tagged proteins can then be used in a simple overnight linkage reaction with the respectively tagged VLPs. Since the modification only happens after the capsomer assembled, obstruction through fused protein is avoided. Furthermore, control of in vivo modification of VLPs via simple induction of the monomers and multiple proteins of interest through ligand based promoters is made possible.

References

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  2. ↑ Ludwig, Christine, and Ralf Wagner. "Virus-like particles—universal molecular toolboxes." Current opinion in biotechnology 18.6 (2007): 537-545. [2]
  3. ↑ Zeltins, Andris. "Construction and characterization of virus-like particles: a review." Molecular biotechnology 53.1 (2013): 92-107. [3]
  4. ↑ Rohovie, Marcus J., Maya Nagasawa, and James R. Swartz. "Virus‐like particles: Next‐generation nanoparticles for targeted therapeutic delivery." Bioengineering & translational medicine 2.1 (2017): 43-57. [4]
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  9. ↑ Sherwood Casjens and Peter Weigele, DNA Packaging by Bacteriophage P22, Viral Genome Packaging Machines: Genetics, Structure, and Mechanism, 2005, pp 80- 88 [9]
  10. ↑ Wageningen, iGEM 2012 [10]
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  12. ↑ Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration into the P22 VLP, Chemical Communications, 2013, 49: 10412-10414 [12]
  13. ↑ Wen Jiang, Zongli Li, Zhixian Zhang, Matthew Baker, Peter Prevelige Jr., and Wah Chiu, Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions, Nature Structural Biology, 2003, 10: 131-135 [13]
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  16. ↑ P A Thuman-Commike, B Greene, J A Malinski, J King, and W Chiu, Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures., Biophysical Journal, 1998, 74: 559-568 [16]
  17. ↑ Hill, Dustin Patterson et al., Sortase-Mediated Ligation as a Modular Approach for the Covalent Attachment of Proteins to the Exterior of the Bacteriophage P22 Virus-like Particle, Bioconjugate Chem., 2017, 28: 2114-2124 [17]
  18. ↑ Alison O`Neil, Courtney Reichhardt, Benjamin Johnson, Peter Prevelige, Trevor Douglas,Genetically Programmed In Vivo Packaging of Protein Cargo and Its Controlled Release from Bacteriophage P22, Angewandte Chemie International Edition, 2011, 50: 7425-7428 [18]
  19. ↑ Sebyung Kang, Masaki Uchida, Alison O’Neil, Rui Li, Peter Prevelige and Trevor Douglas, Implementation of P22 Viral Capsids as Nanoplatforms, Biomacromolecules, 2010, 11: 2804–2809 [19]
  20. ↑ Benjamin Schwarz et al. Symmetry Controlled, Genetic Presentation of Bioactive Proteins on the P22 Virus-like Particle Using an External Decoration Protein, acsnano, 2015. Volume 9 No.9: 9134-9147 [20]
  21. ↑ Kathleen W. Clancy, Jeffrey A. Melvin, Dewey G. McCafferty; Sortase Transpeptidases: Insights Into Mechanism, Substrate Specificity, and Inhibition; WileyPeriodicals, Inc. Biopolymers (Pept Sci) 94: 385–396, 2010; pages 385-386 [21]
  22. ↑ ] Matthew L. Bentley, Erin C. Lamb, and Dewey G. McCafferty; Mutagenesis Studies of Substrate Recognition and Catalysisin the Sortase A Transpeptidase from Staphylococcus&nbps;aureus; THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 21; pages 14762-14771 [22]
  23. ↑ Shaynoor Dramsi, Patrick Trieu-Cuot, Hélène Bierne; Sorting sortases: a nomenclature proposal for the various sortases of Gram-positive bacteria; Research in Microbiology 156 (2005) 289–297; pages 289-291 [23]
  24. ↑ Brenda A. Frankel, Ryan G. Kruger, Dana E. Robinson, Neil L. Kelleher, and Dewey G. McCafferty; Staphylococcus aureus Sortase Transpeptidase SrtA: Insight into the Kinetic Mechanism and Evidence for a Reverse Protonation Catalytic Mechanism Biochemistry, Vol. 44, No. 33, 2005; page 11194 [24]
  25. ↑ Tsutomu Tanaka, Teruyasu Yamamoto, Shinya Tsukiji and Teruyuki Nagamune; Site-Specific Protein Modification on Living Cells Catalyzed by Sortase; ChemBioChem2008, 9, pages 802–807 [25]
  26. ↑ Maximilian Wei-Lin Popp and Hidde L. Ploegh; Making and Breaking Peptide Bonds: ProteinEngineering Using Sortase; Angewandte Chemie international Version 50; 27.04.2011; pages 5024-5026 [26]
  27. ↑ Karin Strijbis, Eric Spooner and Hidde L. Ploegh; Protein Ligation in Living Cells Using Sortase; Traffic 2012 13. edition; page 787 [27]
  28. ↑ Zhimeng Wu, Haofei Hong, Xinrui Zhao and Xun Wang; Efficient expression of Sortase A from Staphylococcus aureus in Escherichia coli and its enzymatic characterizations; Bioresources and Bioprocessing; 18.02.2017; pages 7&8 [28]
  29. ↑ Hee-Jin Jeong, Gita C. Abhiraman, Craig M. Story, Jessica R. Ingram, Stephanie K. Dougan; Generation of Ca2+-independent sortase A mutants with enhanced activity for protein and cell surface labeling; OnePlos 12; 4.12.2017; pages: 2-3 [29]
  30. ↑ Glasgow JE, Salit ML, Cochran JR (2016) In vivo site-specific protein tagging with diverse amines using an engineered sortase variant. J Am Chem Soc 138: 7496–7499 [30]
  31. ↑ Thomas Proft; Sortase-mediated protein ligation: an emerging biotechnology tool for protein modificationand immobilization; Biotechnol Lett (2010) 32; page 9 [31]
  32. ↑ Xueqing Guo, Qianli Wang, Benjamin M. Swarts, and Zhongwu Guo; Sortase-Catalyzed Peptide-Glycosylphosphatidylinositol Analogue Ligation; Journal of the American Chemical Society 2009 131 (29); 9878 [32]
  33. ↑ Yujie Maab, Roeland J.M: Nolteb, Jeroen J.L.M. Cornelissen, Virus-based nanocarriers for drug delivery, Advanced Drug Delivery Reviews,2012, Volume 64: 811-825 [33]
  34. ↑ Roldão A, Mellado MC, Castilho LR, Carrondo MJ, Alves PM, Virus-like particles in vaccine development., Expert Rev Vaccines, 2010, 9: 1149-1176 [34]
  35. ↑ Schwarz B, Morabito KM, Ruckwardt TJ, Patterson DP, Avera J, Miettinen HM, Graham BS, Douglas T, Virus-like Particles Encapsidating Respiratory Syncytial Virus M and M2 Proteins Induce Robust T Cell Responses, ACS Biomater Sci Eng. 2016; 2(12): 2324–2332 [35]
  36. ↑ Molino NM, Wang S Caged Protein nanoparticles for drug delivery, Cur. Opinion in Biotechnology, 2014; 28: 75-82 [36]
  37. ↑ Sebyung Kang Gabriel C. Lander John E. Johnson Prof. Peter E. Prevelige Prof., Development of Bacteriophage P22 as a Platform for Molecular Display: Genetic and Chemical Modifications of the Procapsid Exterior Surface, ChemBioChem, 2008, 9(4): 514-518 [37]
  38. ↑ Sebyung Kang Gabriel C. Lander John E. Johnson Prof. Peter E. Prevelige Prof., Development of Bacteriophage P22 as a Platform for Molecular Display: Genetic and Chemical Modifications of the Procapsid Exterior Surface, ChemBioChem, 2008, 9(4): 514-518 ↑ RV Mannige, CL Brooks, Periodic Table of Virus Capsids: Implications for Natural Selection and Design, PLoS One, 2010; 5(3): e9423 [37] ">[38]
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