Protein engineering through chemical, genetic and computational manipulation

Itaru Hamachi *ab and Gonçalo J. L. Bernardes *cd
aDepartment of Synthetic Chemistry and Biological Chemistry, Kyoto University, Kyoto, Japan. E-mail:
bCore Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo, Japan
cInstituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Portugal
dDepartment of Chemistry, University of Cambridge, Cambridge, UK. E-mail:

Received 13th November 2018
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Itaru Hamachi

Itaru Hamachi obtained his PhD at the Department of Synthetic Chemistry of Kyoto University in 1988 under the supervision of Prof. I. Tabushi. He started his academic career as an assistant professor in Prof. T. Kunitake's group at Kyushu University in 1988, and then moved to S. Shinkai's lab in the same department as an associate professor. In 2001, he became a full professor there and returned back to Kyoto University (Department of Synthetic Chemistry and Biological Chemistry) in 2005. He is also now a research director of the ERATO project supported by JST. His interest has recently been in chemical biology, organic chemistry in multimolecular crowding biosystems, and supramolecular multicomponent systems.

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Gonçalo J. L. Bernardes

Gonçalo Bernardes, after completing his DPhil degree in 2008 at the University of Oxford, UK, undertook postdoctoral work at the Max-Planck Institute of Colloids and Interfaces, Germany, and ETH Zürich, Switzerland, and worked as a Group Leader at Alfama Lda in Portugal. He started his independent research career in 2013 at the University of Cambridge as a Royal Society University Research Fellow, and in 2018 he was appointed as University Lecturer. He is also the Director of the Chemical Biology unit at iMM Lisboa. In 2016, Gonçalo's research was recognised with the award of the Chem. Soc. Rev. Emerging Investigator Lectureship. His research group interests focus on the use of chemistry principles to tackle challenging biological problems for understanding and fighting cancer.

Biocomplexity—the study of complex structures and behaviours that take place in biological systems—goes beyond direct protein production from genes to higher orders of complexity that can be added after a gene has been translated into a protein. These processes can significantly expand the functionality of a protein by adding specific chemical groups to specific sites. The ability to use chemical and enzymatic methods to introduce specific groups into folded proteins is now a powerful strategy to add functional capacity to a protein. This strategy can help to elucidate the role of such post-translational groups on a protein and promises protein therapeutics and glycoconjugate vaccines with optimal properties. Furthermore, a number of methods have been developed towards the total synthesis of proteins. These methods enable the synthesis of complex proteins that have oligosaccharides at pre-defined positions, which cannot be achieved by other means. The sequence of proteins can also be evolved by directed evolution, in order to catalyse the formation of even non-natural bonds, or computationally to design a sequence that has high affinity and specificity for a complementary sequence present on an antigen of interest. This themed issue highlights different approaches by which a protein's structure and functional capacity can be explored, either by adding functional groups or by evolving the native sequence. These approaches may be used to elucidate the role of naturally occurring post-translational modifications, or to create protein conjugates as therapeutics or vaccines. This issue also includes reviews on synthetic antibodies by computational design and the use of proteins as building blocks to create nanostructures.

Directed evolution of enzymes uses our capacity to take enzymes found in nature and by applying design principles of chemical reactivity their DNA blueprint may be tweaked to improve their catalytic capacity or—even more excitingly—to catalyse the creation of a new bond. For her work on directed evolution of enzymes, Frances Arnold has been awarded the Nobel Prize in Chemistry 2018. In her review, together with co-workers, she describes how enzyme engineering strategies can be applied to amino acid synthases to produce artificial synthases capable of producing amino acids beyond the twenty canonical ones. The generation of these synthases enables the efficient and environmentally friendly production of a wide variety of non-canonical amino acids that have non-natural side-chains with different chemical properties and reactivities (DOI: 10.1039/C8CS00665B). Combined with genetic encoding methods, these side-chains may add new structural or functional features into designer proteins.

The introduction of specific groups, such as fluorophores or cytotoxic drugs, at pre-determined sites on a protein of interest offers ways to track and follow their uptake and intracellular trafficking and to precisely deliver a potent anti-cancer drug into a tumour while sparing healthy tissues. The use of enzymes to achieve site-specific labelling of proteins offers advantages, such as specificity and conjugation efficiency. Distefano and co-workers start by reviewing key methods developed in the last five years to chemically install modifications on proteins either in the test tube or bioorthogonally in cells and even organisms. The authors then detail advances in enzymatic protein labelling in four sections that describe methods based on peptidases, transferases, ligases, and oxidoreductases. Methods based on enzymes typically achieve high specificity for the intended site and reaction conversion efficiencies (DOI: 10.1039/C8CS00537K). Equally, the chemical covalent attachment of polymers, such as polyethylene glycol, to therapeutic proteins is a successful strategy to modulate a protein's pharmacokinetic properties (i.e., extension of half-life in circulation) and reduce potential immunogenicity. Maynard and Ko report on advances in the synthetic design of polymers for protein modulation and strategies to achieve site-specific modification. The site and polymer-to-protein ratio have been shown to dramatically influence the protein's activity and properties, thus methods that allow attachment of the polymer at defined sites are increasingly important (DOI: 10.1039/C8CS00606G).

Successful vaccine design often requires the covalent linkage of oligosaccharide antigens, which are poorly immunogenic, into protein carriers that are highly immunogenic. Adamo and Berti describe how antimicrobial glycoconjugate vaccine design has evolved from more classical approaches that used heterogeneous mixtures into new designs that place a focus on synthetic purity of the oligosaccharide as well as the final glycoprotein vaccine construct. This focus on chemical precision and purity is enabling further insights at the molecular level on the mode of action of glycoconjugate vaccines and hopefully will lead to more efficient vaccines in the future (DOI: 10.1039/C8CS00495A).

As an alternative to post-translational chemical modification of proteins, chemists have and continue to advance efforts to apply synthetic logic into the assembly of proteins. In this context, native and expressed chemical ligation enables the chemo- and regio-selective formation of a native amide bond between unprotected polypeptides from synthetic and recombinant origins. The application of this strategy now allows construction of proteins of moderate size that have, for example, defined post-translational modifications installed through synthesis at pre-defined sites. This technique is extremely powerful and recent advances in the field of chemical ligation are discussed by Payne, Becker and co-workers in their review that includes examples in which such approaches have generated post-translationally modified proteins that cannot be obtained by genetic means (DOI: 10.1039/C8CS00573G).

Instead of conjugating synthetic groups at a site of the sequence of a protein to increase functional capacity, Weil and co-workers describe the use of proteins as building blocks in combination with site-selective chemical reactions to build precise protein-based nanostructures. Particular emphasis is placed on the design of protein building blocks, recognition units and linkers, and their subsequent assembly into structurally defined, functional protein nanostructures for applications in catalysis, materials and biomedical sciences (DOI: 10.1039/C8CS00590G).

Computational methods play an increasingly important role in the de novo design of proteins as well as in precise sequence alterations to modulate their properties and stability. The in silico rational design of antibodies specific to a disease-relevant antigen is referred to as the third-generation method by Vendruscolo and co-workers in their review. This third-generation approach is the next logical step in antibody discovery after in vivo (first generation) and in vitro (second generation) approaches (DOI: 10.1039/C8CS00523K). This review provides an in-depth discussion of current approaches for de novo protein design and includes examples of when their use has dramatically accelerated discoveries in the biomedical field. In a second example of the power of computational methods for protein engineering, Kazlauskas describes the development of web-based tools that enable the identification of amino acid replacements or additions to a protein's original sequence to fine-tune their stability and, thus, functional state. For example, destabilization of the unfolded form of a protein by addition of a disulfide linkage or through introduction of prolines may lead to stabilization of the folded form. Other sources of potential protein destabilization, such as oxidation, proteolytic cleavage or aggregation of partially unfolded states are also discussed along with ways on how to prevent these (DOI: 10.1039/C8CS00014J).

In conclusion, this themed issue shows that methods for engineering proteins, the workhorses of biology, provide many opportunities to develop interdisciplinary approaches that range from computational to synthetic to biochemical. Given the great progress in protein engineering, we are being equipped with valuable methods for modifying and manipulating molecularly complicated proteins and are now ready to go further toward new directions. Systems design of an artificial protein/enzyme network by using a set of well-controlled engineered proteins would create novel intelligent soft-matter and sophisticated biomaterials in synthetic biology, which would also give some insight for a design principle of proto-cells. Elaborately invading or controlling a natural protein/gene network with engineered proteins should afford new discoveries in a variety of fields of biology/medicine, that could have substantial impact on chemical biology and lead to new biopharmaceutical applications beyond the conventional vaccine and protein therapeutics. This themed issue clearly suggests that development of non-canonical molecules and strategies are keenly desirable in order to evoke new functions of proteins beyond natural systems. It is now the era for challenging the higher level of biocomplexity using integrated chemistry.

This journal is © The Royal Society of Chemistry 2018