Self‐renaturing enzymes: Design of an enzyme‐chaperone chimera as a new approach to enzyme stabilization

Bergeron, Lisa M.; Gomez, Lizabeth; Whitehead, Timothy A.; Clark, Douglas S.

doi:10.1002/bit.22254

Cited by 37 publications

(19 citation statements)

References 24 publications

(27 reference statements)

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“…Reducing linker length by mutation to within 5–10 amino acids can alter both the activity and stability of the N- and C-terminal functional domains 50,51. Conversely, increasing linker length to 60–70 amino acids may render the functional domains insensitive to one another 52,53.…”

Section: Discussionmentioning

confidence: 99%

Internal Regulatory Interactions Determine DNA Binding Specificity by a Hox Transcription Factor

Liu

Matthews

Bondos

2009

Journal of Molecular Biology

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SummaryIn developing bilaterans, the Hox transcription factor family regulates batteries of downstream genes to diversify serially repeated units. Given Hox homeodomains bind a wider array of DNA binding sites in vitro than are regulated by the full-length protein in vivo, regions outside the homeodomain must aid DNA site selection. Indeed, we find affinity for disparate DNA sequences varies less than 3-fold for the homeodomain isolated from the Drosophila Hox protein Ultrabithorax Ia (UbxHD), whereas for the full-length protein (UbxIa) affinity differs by more than 10-fold. The rank order of preferred DNA sequences also differs, further demonstrating distinct DNA binding preferences. The increased specificity of UbxIa can be partially attributed to the I1 region, which lies adjacent to the homeodomain and directly impacts binding energetics. Each of three segments within I1 -the Extradenticle-binding YPWM motif, the 6 amino acids immediately N-terminal to this motif, and the 8 amino acids abutting the YPWM C-terminus -uniquely contribute to DNA specificity. Combination of these regions synergistically modifies DNA binding to further enhance specificity. Intriguingly, the presence of the YPWM motif in UbxIa inhibits DNA binding only to Ubx•Extradenticle heterodimer binding sites, potentially functioning in vivo to prevent Ubx monomers from binding and misregulating heterodimer target genes. However, removal of the surrounding region allows the YPWM motif to also inhibit binding to Hox-only recognition sequences. Despite a modular domain design for Hox proteins, these results suggest that multiple Hox protein regions form a network of regulatory interactions that coordinate context-and genespecific responses. Since most non-homeodomain regions are not conserved between Hox family members, these regulatory interactions have the potential to diversify binding by the highly homologous Hox homeodomains.

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Section: Discussionmentioning

confidence: 99%

Internal Regulatory Interactions Determine DNA Binding Specificity by a Hox Transcription Factor

Liu

Matthews

Bondos

2009

Journal of Molecular Biology

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“…In a new focus to the optimization of the enzyme properties for this reaction, Clark and coworkers performed the refolding of a PGA -chaperone chimera under certain conditions where the enzyme properties appeared to be optimal, improving the performance of the reactivated PGA in kinetically controlled synthesis [160].…”

Section: Effect Of the Immobilization Protocol On The Enzyme Propertiesmentioning

confidence: 99%

Use of Enzymes in the Production of Semi-Synthetic Penicillins and Cephalosporins: Drawbacks and Perspectives

Volpato¹,

Rodrigues

Fernández‐Lafuente

2010

CMC

112

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Semi-synthetic β-lactamic antibiotics are the most used anti-bacteria agents, produced in hundreds tons/year scale. It may be assumed that this situation will even increase during the next years, with new β-lactamic antibiotics under development. They are usually produced by the hydrolysis of natural antibiotics (penicillin G or cephalosporin C) and the further amidation of natural or modified antibiotic nuclei with different carboxylic acyl donor chains. Due to the contaminant reagents used in conventional chemical route, as well as the high energetic consumption, biocatalytic approaches have been studied for both steps in the production of these very interesting medicaments during the last decades. Recent successes in some of these methodologies may produce some significant advances in the antibiotics industry. In fact, the hydrolysis of penicillin G to produce 6-APA catalyzed by penicillin G acylase is one of the most successful historical examples of the enzymatic biocatalysis, and much effort has been devoted to find enzymatic routes to hydrolyze cephalosporin C. Initially this could be accomplished in a quite complex system, using a two enzyme system (D-amino acid oxidase plus glutaryl acylase), but very recently an efficient cephalosporin acylase has been designed by genetic tools. Other strategies, including metabolic engineering to produce other antibiotic nuclei, have been also reported. Regarding the amidation step, much effort has been devoted to the improvement of penicillin acylases for these reactions since 1960. New reaction strategies, continuous product extraction or new penicillin acylases with better properties have proven to be the key to have competitive biocatalytic processes. In this review, a critical discussion of these very interesting advances in the application of enzymes for the industrial synthesis of semi-synthetic antibiotics will be presented.

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“…Complementary oligonucleotide pairs of 15,20,30,35,40, and 45 nucleotides that do not form secondary structures and have stable hybridization at 37 C were designed using NUPACK (34) (see Table S1 for the oligonucleotide sequences). The 5 0 end of the oligonucleotides was first modified with a primary amine via a C 6 linker (purchased from Integrated DNA Technologies, Coralville, IA).…”

Section: Bg-dna Conjugationmentioning

confidence: 99%

“…The length and flexibility of the linker have a significant impact on the accessibility of the antibody and cytokine to their respective binding targets. For example, a sufficient separation between the two protein domains is necessary for optimal fusion protein activity (12)(13)(14)(15)(16)(17)(18)(19). Moreover, rigid linkers often result in better protein domain separation and fusion protein activity compared with flexible linkers (12)(13)(14)(15)(16)(17)(18)(19).…”

Section: Introductionmentioning

confidence: 98%

Designing Cell-Targeted Therapeutic Proteins Reveals the Interplay between Domain Connectivity and Cell Binding

Robinson-Mosher¹,

Chen

Way³

et al. 2014

Biophysical Journal

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The therapeutic efficacy of cytokines is often hampered by severe side effects due to their undesired binding to healthy cells. One strategy for overcoming this obstacle is to tether cytokines to antibodies or antibody fragments for targeted cell delivery. However, how to modulate the geometric configuration and relative binding affinity of the two domains for optimal activity remains an outstanding question. As a result, many antibody-cytokine complexes do not achieve the desired level of cell-targeted binding and activity. Here, we address these design issues by developing a computational model to simulate the dynamics and binding kinetics of natural and engineered fusion proteins such as antibody-cytokine complexes. To verify the model, we developed a modular system in which an antibody fragment and a cytokine are conjugated via a DNA linker that allows for programmable linker geometry and protein spatial configuration. By assembling and testing several anti-CD20 antibody fragment-interferon ? complexes, we showed that varying the linker length and cytokine binding affinity controlled the magnitude of cell-targeted signaling activation in a manner that agreed with the model predictions, which were expressed as dose-signaling response curves. The simulation results also revealed that there is a range of cytokine binding affinities that would achieve optimal therapeutic efficacy. This rapid prototyping platform will facilitate the rational design of antibody-cytokine complexes for improved therapeutic outcomes.

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Self‐renaturing enzymes: Design of an enzyme‐chaperone chimera as a new approach to enzyme stabilization

Cited by 37 publications

References 24 publications

Internal Regulatory Interactions Determine DNA Binding Specificity by a Hox Transcription Factor

Internal Regulatory Interactions Determine DNA Binding Specificity by a Hox Transcription Factor

Use of Enzymes in the Production of Semi-Synthetic Penicillins and Cephalosporins: Drawbacks and Perspectives

Designing Cell-Targeted Therapeutic Proteins Reveals the Interplay between Domain Connectivity and Cell Binding

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