Strategies for Increasing Protein Stability

Chandler, Peter G.; Broendum, Sebastian S.; Riley, Blake T.; Spence, Matthew A.; Jackson, Colin J.; McGowan, Sheena; Buckle, Ashley M.

doi:10.1007/978-1-4939-9869-2_10

Cited by 26 publications

(29 citation statements)

References 109 publications

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“…There is a historic body of work related to rationally grafting CDR loops between antibodies (49). In practice, this grafting technique involves a balance of transferring residues involved in binding and removing stabilizing residues in the accepting scaffold (18,50), which may reveal certain pairs of donor and recipient scaffolds to be graft-incompatible (36). The Adnectin-anti-VEGFR2 binding loops were compatible with the FN3Con scaffold, given the almost-complete transfer of affinity.…”

Section: Discussionmentioning

confidence: 99%

“…There are multiple approaches of improving the stability of protein folds (18). Protein stability exists between two critical thresholds, where a protein is able to fold into a stable, native 3-dimensional shape but is also still dynamic enough to perform its functions.…”

Section: Introductionmentioning

confidence: 99%

“…Multiple, well-established techniques use phylogenetic information to derive thermostable homologs for a given protein family (18,(30)(31)(32)(33)(34). Here we have specifically applied consensus design, which identifies conserved residues within a protein family that are likely to be stabilizing (31,34).…”

Section: Introductionmentioning

confidence: 99%

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Mutational and biophysical robustness in a pre-stabilized monobody

Chandler

Tan

Porebski

et al. 2020

Preprint

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The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyper-stable monobody derivative with diagnostic and therapeutic potential. Pre-stabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain towards biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerisation. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the pre-stabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a pre-stabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics, and is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Mutational and biophysical robustness in a pre-stabilized monobody

Chandler

Tan

Porebski

et al. 2020

Preprint

Self Cite

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“…This Tenascin-FN3 derived monobody is now under clinical development as 'Tn3 by Viela Bio (spun out from AstraZeneca's Medimmune) [31,32]. Finally, in order to design more robust monobodies with substantially higher initial stabilities, the consensus bioinformatic technique [33] has been applied to generate Aro Therapeutic's Centyrins from a consensus sequence of 14 FN3 domains (spun out from Janssen) [34,35] and the hyper-stable "Consensus FN3" FN3Con domain [36] from a consensus of 2123 FN3 sequences. As a result of this differentiation, these synthetic domains share the parent FN3 fold but feature varying sequence similarity ( Figure 1D,E).…”

Section: Modern Fn3 Derivatives In Developmentmentioning

confidence: 99%

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

Chandler

Buckle

2020

Cells

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As a non-antibody scaffold, monobodies based on the fibronectin type III (FN3) domain overcome antibody size and complexity while maintaining analogous binding loops. However, antibodies and their derivatives remain the gold standard for the design of new therapeutics. In response, clinical-stage therapeutic proteins based on the FN3 domain are beginning to use native fibronectin function as a point of differentiation. The small and simple structure of monomeric monobodies confers increased tissue distribution and reduced half-life, whilst the absence of disulphide bonds improves stability in cytosolic environments. Where multi-specificity is challenging with an antibody format that is prone to mis-pairing between chains, multiple FN3 domains in the fibronectin assembly already interact with a large number of molecules. As such, multiple monobodies engineered for interaction with therapeutic targets are being combined in a similar beads-on-a-string assembly which improves both efficacy and pharmacokinetics. Furthermore, full length fibronectin is able to fold into multiple conformations as part of its natural function and a greater understanding of how mechanical forces allow for the transition between states will lead to advanced applications that truly differentiate the FN3 domain as a therapeutic scaffold.

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“…Based on the analysis of the currently existing phylogenic descendants ADH1 (ethanol generation) and ADH2 (ethanol consumption), an inferred and constructed ancestral ADH had the functions of both descendants. 45 ASR can be used to design mutants with a higher degree of promiscuity and less specificity than the currently existing enzymes, 46 to engineer the thermostability of proteins, 47,48 and to elucidate the molecular mechanism of enzymes. 49,50 A limited number of mutations at the amino acid level can cause significant differences in protein properties, which is also the case of naturally existing ancestral transcription factor PmrA.…”

Section: Employing Compatible Heterogeneous Tfsmentioning

confidence: 99%

Transcriptional factor engineering in microbes for industrial biotechnology

Wang

Liang

Xing

et al. 2020

J of Chemical Tech & Biotech

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Transcription can be regulated by controlling the provision of transcriptional factors (TFs), because TFs determine the transcription process by specifying the binding of RNA polymerase holoenzyme on promoters. Manipulations, such as replacing endogenous TFs with mutants obtained through directed evolution (or rational design), and introducing compatible heterogeneous TFs, are effective ways to regulate transcription. Designing artificial TFs with desired properties by following the principles of protein design and reconstructing ancestral TFs with the information gained from a perspective of evolution by multiple sequence alignments of the related TFs, are feasible alternative options. The engineered TFs can be used to create inducible protein expression systems that respond to a specific stimulus. Moreover, the engineered TFs could lead to a transcriptional profile different from that of the wild-type strains. Accordingly, these engineered TFs could be utilized to construct strains resistant to adverse conditions, since the emergence of resistance generally requires global transcriptional changes at the transcriptomic scale. Meanwhile, these engineered TFs can also be employed in the construction of sophisticatedly designed genetic circuits for diverse purposes. In this paper, we reviewed the advances in the above-mentioned aspects.

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Strategies for Increasing Protein Stability

Cited by 26 publications

References 109 publications

Mutational and biophysical robustness in a pre-stabilized monobody

Mutational and biophysical robustness in a pre-stabilized monobody

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

Transcriptional factor engineering in microbes for industrial biotechnology

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