In vitro Fab display: a cell-free system for IgG discovery

Stafford, Ryan L.; Matsumoto, Marissa L.; Yin, Gang; Cai, Qianqian; Fung, Juan José; Stephenson, Heather; Gill, Avinash; You, Monica; Lin, Shwu-Hwa; Wang, Willie D.; Masikat, Mary Rose; Li, Xiaofan; Penta, Kalyani; Steiner, Alex; Baliga, Ramesh; Murray, Christopher J L; Thanos, Christopher D.; Hallam, Trevor J.; Sato, Aaron K.

doi:10.1093/protein/gzu002

Cited by 37 publications

(28 citation statements)

References 42 publications

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“…However, the emulsion step reduced the library complexity to a similar range as in vivo methods. To achieve an in vitro selection of multi-subunit proteins of potentially up to 10 14 variants, ribosome display was performed where one Fab subunit was randomized while the other subunit was held constant [49•]. This was carried out with both heavy chain and light chain libraries yielding tight binders to VEGF and CEA.…”

Section: Expanding the Scope Of Selections To New Propertiesmentioning

confidence: 99%

Advances in the directed evolution of proteins

Lane

Seelig

2014

Current Opinion in Chemical Biology

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Natural evolution has produced a great diversity of proteins that can be harnessed for numerous applications in biotechnology and pharmaceutical science. Commonly, specific applications require proteins to be tailored by protein engineering. Directed evolution is a type of protein engineering that yields proteins with the desired properties under well-defined conditions and in a practical time frame. While directed evolution has been employed for decades, recent creative developments enable the generation of proteins with previously inaccessible properties. Novel selection strategies, faster techniques, the inclusion of unnatural amino acids or modifications, and the symbiosis of rational design approaches and directed evolution continue to advance protein engineering.

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Section: Expanding the Scope Of Selections To New Propertiesmentioning

confidence: 99%

Advances in the directed evolution of proteins

Lane

Seelig

2014

Current Opinion in Chemical Biology

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“…For example, the scFab scaffold greatly simplifies antibody reformatting to scIgG or fusion to other tags and thus will aid in efficient downstream antibody validation. Reformatting lead scFabs for an in vitro transcription-translation format capable of high-level IgG production 38, 39 could yield purified scIgG in only one day compared to more than one week for mammalian cell production. Ultimately, this scFab format will help improve the generation of novel antibodies from recombinant antibody libraries to advance biomedical research and therapeutics.…”

Section: Discussionmentioning

confidence: 99%

An Improved Single-Chain Fab Platform for Efficient Display and Recombinant Expression

Koerber

Hornsby

Wells

2015

Journal of Molecular Biology

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Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study proteins and their many post-translational modification states; however many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain Fab (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain Fv with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1–3 mg per liter. Furthermore, rerouting of the scFab to the co-translational signal recognition particle (SRP) pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable (81°C Tm), predominantly monomeric (>90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies.

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“…This strategy is costly, inefficient and prone with challenges. A far more powerful and efficient approach is in vitro selection of Fab fragments using phage display [202,203] or, less often, ribosome display [204,205]. Multiple templates have been used, but the most common is that based on the herceptin scaffold.…”

Section: Target Protein Modification For Enhanced Crystallizabilitymentioning

confidence: 99%

The “Sticky Patch” Model of Crystallization and Modification of Proteins for Enhanced Crystallizability

Derewenda

Godzik

2017

Methods in Molecular Biology

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Crystallization of macromolecules has long been perceived as a stochastic process, which cannot be predicted or controlled. This is consistent with another popular notion that the interactions of molecules within the crystal, i.e. crystal contacts, are essentially random and devoid of specific physicochemical features. In contrast, functionally relevant surfaces, such as oligomerization interfaces and specific protein-protein interaction sites, are under evolutionary pressures so their amino acid composition, structure and topology are distinct. However, current theoretical and experimental studies are significantly changing our understanding of the nature of crystallization. The increasingly popular ‘sticky patch’ model, derived from soft matter physics, describes crystallization as a process driven by interactions between select, specific surface patches, with properties thermodynamically favorable for cohesive interactions. Independent support for this model comes from various sources including structural studies and bioinformatics. Proteins that are recalcitrant to crystallization can be modified for enhanced crystallizability through chemical or mutational modification of their surface to effectively engineer ‘sticky patches’ which would drive crystallization. Here, we discuss the current state of knowledge of the relationship between the microscopic properties of the target macromolecule and its crystallizability, focusing on the ‘sticky patch’ model. We discuss state-of-art in silico methods that evaluate the propensity of a given target protein to form crystals based on these relationships, with the objective to design of variants with modified molecular surface properties and enhanced crystallization propensity. We illustrate this discussion with specific cases where these approaches allowed to generate crystals suitable for structural analysis.

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In vitro Fab display: a cell-free system for IgG discovery

Cited by 37 publications

References 42 publications

Advances in the directed evolution of proteins

Advances in the directed evolution of proteins

An Improved Single-Chain Fab Platform for Efficient Display and Recombinant Expression

The “Sticky Patch” Model of Crystallization and Modification of Proteins for Enhanced Crystallizability

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